Novel Use of Spiegelmers

ABSTRACT

The present invention relates to the use of a L-nucleic acid as intracellularly active agent.

One aspect of the present invention relates to a new use of spiegelmers.Another aspect of the present invention relates to spiegelmers that bindHMG proteins.

With advances in molecular medicine it has become possible to identifytarget molecules involved in a disease or a disease state and to act onthese specifically so as thereby to treat or prevent the disease or thedisease state or at least to alleviate the symptoms associatedtherewith. The target molecules can in principle be divided into twogroups. A first group includes target molecules that are presentextracellularly and can thus in principle be brought into contact withan active substance by administering the latter in a body fluid or abody cavity that contains the target molecule. The first group of targetmolecules is herein also referred to as extracellular target molecules.The second group of target molecules includes target molecules that arepresent in cells, these cells being involved in the disease to betreated or in the predisposition to the disease. It is not necessary inthis connection for the target molecule to be directly responsible forthe disease state or directly connected with the predisposition to thedisease. Instead, it is sufficient if the respective target molecule isinvolved in an action cascade, the course of which is influenced by theactive substance, with the result that the active substance is suitablefor the treatment or prevention of the disease. The second group oftarget molecules is herein also referred to as intracellular targetmolecules.

The nature of the target molecule, i.e. extracellular or intracellulartarget molecule, determines in principle the binding class, with whichan attempt can be made to effect the interaction, necessary for thetherapeutic or preventive action, between the active substance,typically the pharmaceutical active substance, and the target molecule.In virtually all cases so-called small molecules can be used, i.e.chemical compounds with a molecular weight of typically 1000 daltons orless. These molecules can interact in the desired manner directly withextracellular target molecules, as well as with intracellular targetmolecules.

Against this background new classes of active substances have beendeveloped by the biotechnology industry, such as for example antibodies,in particular monoclonal antibodies, antisense molecules, siRNAmolecules, aptamers and spiegelmers. Although some of these classes ofmolecules are still in the preliminary stage of clinical investigations,there exist at least in the case of antibodies and antisense moleculesproducts that are already in clinical use. However, with these newclasses of substances there are also significant problems as regardsaddressing intracellular target molecules. Thus, for example, theintracellular use of antibodies is currently still not always possible,at least not to an extent or in a way and manner that allows a routineuse in patients of antibodies directed against intracellular targetmolecules for the purposes of treatment and/or prophylaxis. Also, theother new classes of active substances, in particular antisensemolecules and siRNA molecules, must on account of their action mechanismbe introduced into the respective cell containing the target molecule orthe gene coding for the target molecule. The targeted release of theactive substance, also termed delivery, is also for these classes ofsubstances the currently limiting factor for a clinical application.

The same is also true of aptamers and spiegelmers, i.e. functionalnucleic acids with a defined three-dimensional structure that allows thespecific interaction with the respective target molecules. The use ofaptamers in order to address intracellular target molecules utilisesmethods of gene technology, more specifically gene therapy. Theaptamers, also termed intramers, directed against an intracellulartarget molecule are incorporated into the respective target cell bymeans of gene technology methods. Such an approach is however alsosubject to considerable limitations, not least on account of the lack ofacceptance of treatment approaches based on gene therapy. In particularthe route adopted in the case of intramers, involving intracellularexpression of a nucleic acid coding intracellularly for the respectiveaptamer, is in principle closed to spiegelmers, since no biologicalsystem exists which would be capable of synthesising spiegelmers, i.e.aptamers consisting of L-nucleotides.

The object of the present invention is accordingly to provide a class ofsubstances that is able to interact specifically with intracellulartarget molecules, i.e. target molecules that are present in a cell.

According to the invention this object is achieved by the subject-matterof the accompanying independent claims. Preferred embodiments aredisclosed in the sub-claims.

According to the present invention the basic object is achieved by thesubject-matter of the independent claims. Preferred embodiments aredisclosed in the sub-claims.

According to a first aspect of the invention the object is achieved bythe use of a L-nucleic acid as intracellular active agent.

In a first embodiment of the first aspect the L-nucleic acid is aspiegelmer.

In a second embodiment of the first aspect, which is also an embodimentof the first embodiment, the L-nucleic acid interacts with anintracellular receptor.

In a third embodiment of the first aspect, which is also an embodimentof the second embodiment, the intracellular receptor is selected fromthe group comprising molecular receptors, enzymes, chaperone molecules,signal peptides, intracellular structures and metabolic intermediates.

In a fourth embodiment of the first aspect, which is also an embodimentof the second embodiment, the intracellular receptor is selected fromthe group comprising polypeptides, carbohydrates, nucleic acids, lipidsand combinations thereof.

In a fifth embodiment of the first aspect, which is also an embodimentof the second, third and fourth embodiment, the L-nucleic acid interactswith an intracellular receptor within a cell.

In a sixth embodiment of the first aspect, which is also an embodimentof the second, third, fourth and fifth embodiment, the intracellularreceptor is selected from the group comprising transcription factors andDNA-binding proteins binding an AT hook.

In a seventh embodiment of the first aspect, which is also an embodimentof the sixth embodiment, the intracellular receptor is selected from thegroup comprising HMG proteins, preferably from the group comprisingHMGA1, HMGA1a, HMGA1b, and HMGA2.

According to a second aspect of the present invention this object isachieved by a method for binding an intracellular receptor, comprising:

-   -   providing a cell containing at least one intracellular receptor,    -   providing a L-nucleic acid, and    -   incubating the cell with the L-nucleic acid.

In a first embodiment of the second aspect the incubation takes placeunder conditions so that the L-nucleic acid binds to the intracellularreceptor in the cell.

In a second embodiment of the second aspect, which is also an embodimentof the first embodiment, the L-nucleic acid is a spiegelmer.

In a third embodiment of the second aspect, which is also an embodimentof the first and second embodiment, after the incubation of the cellwith the L-nucleic acid it is determined whether a binding, inparticular an intracellular binding, of the L-nucleic acid to theintracellular receptor has taken place.

In a fourth embodiment of the second aspect, which is also an embodimentof the first, second and third embodiment, the intracellular receptor isselected from the group comprising molecular receptors, metabolicintermediates and enzymes.

In a fifth embodiment of the second aspect, which is also an embodimentof the first, second, third and fourth embodiment, the intracellularreceptor is selected from the group comprising polypeptides,carbohydrates, nucleic acids, lipids and combinations thereof.

In a sixth embodiment of the second aspect, which is also an embodimentof the first, second, third, fourth and fifth embodiment, theintracellular receptor is selected from the group comprisingtranscription factors and DNA-binding proteins binding an AT hook.

In a seventh embodiment of the second aspect, which is also anembodiment of the sixth embodiment, the intracellular receptor isselected from the group comprising HMG proteins, and is preferablyselected from the group comprising HMGA1, HMGA1a, HMGA1b and HMGA2.

According to a third aspect of the invention this object is achieved byuse of a L-nucleic acid to manufacture a medicament for the treatmentand/or prevention of a disease, the target molecule of the medicamentbeing an intracellular target molecule.

In a first embodiment of the third aspect the intracellular receptor isselected from the group comprising molecular receptors, enzymes,chaperone molecules, signal peptides, intracellular structures andmetabolic intermediates.

In a second embodiment of the third aspect, which is also an embodimentof the first embodiment, the intracellular receptor is selected from thegroup comprising polypeptides, carbohydrates, nucleic acids, lipids andcombinations thereof.

In a third embodiment of the third aspect, which is also an embodimentof the first and second embodiment, the target molecule is selected fromthe group comprising transcription factors and DNA-binding proteinsbinding an AT hook.

In a fourth embodiment of the third aspect, which is also an embodimentof the third embodiment, the target molecule is selected from the groupcomprising HMG proteins, and is preferably selected from the groupcomprising HMGA1, HMGA1a, HMGA1b and HMGA2.

In a fifth embodiment of the third aspect, which is also an embodimentof the third and fourth embodiment, the disease is selected from thegroup comprising tumour diseases, virus infections and arteriosclerosis.

In a sixth embodiment of the third aspect, which is also an embodimentof the fifth embodiment, the tumour disease is selected from the groupcomprising mesenchymal tumours, epithelial tumours, benign tumours,malignant tumours and metastasising tumours.

In a seventh embodiment of the third aspect, which is also an embodimentof the third, fourth, fifth and sixth embodiment, the target molecule isHMGA and the diseases are selected from the group comprising carcinomasof the prostate, pancreas, thyroid, cervix, stomach, breast,colon/rectum, ovaries; pneuroblastomas; lymphomas, uterine leiomyomas;lipomas; endometrial polyps; chondroid hamartomas of the lungs;pleomorphic adenomas of the salivary glands; haemangiopericytomas;chondromatous tumours; aggressive angiomyxomas; diffuse astrocytomas;osteoclastomas; skin cancer; Burkitt's lymphoma; Lewis lung cancer;leukaemia; non-small-cell lung cancer; as well as in each casemetastases and/or metastasising forms thereof.

In an eighth embodiment of the third aspect, which is also an embodimentof the fifth embodiment, the arteriosclerosis is triggered or caused byformation of arteriosclerotic plaques mediated by HMGA1, HMGA1a, HMG1band/or HMGA2.

In a ninth embodiment of the third aspect, which is also an embodimentof the first, second, third, fourth, fifth, sixth, seventh and eighthembodiment, the intracellular target molecule is presentintracellularly.

According to a fourth aspect of the invention the object is achieved bythe use of a L-nucleic acid for the manufacture of a diagnostic agentfor diagnostic purposes, the target molecule of the diagnostic agentbeing an intracellular target molecule.

In a first embodiment of the fourth aspect the intracellular receptor isselected from the group comprising molecular receptors, enzymes,chaperone molecules, signal peptides, intracellular structures andmetabolic intermediates.

In a second embodiment of the fourth aspect, which is also an embodimentof the first embodiment, the intracellular receptor is selected from thegroup comprising polypeptides, carbohydrates, nucleic acids, lipids andcombinations thereof.

In a third embodiment of the fourth aspect, which is also an embodimentof the first and second embodiment, the target molecule is selected fromthe group comprising transcription factors and DNA-binding proteinsbinding an AT hook.

In a fourth embodiment of the fourth aspect, which is also an embodimentof the third embodiment, the target molecule is selected from the groupcomprising HMG proteins, and is preferably selected from the groupcomprising HMGA, HMGA1a, HMGA1b and HMGA2.

In a fifth embodiment of the fourth aspect, which is also an embodimentof the third and fourth embodiment, the disease is selected from thegroup comprising tumour diseases, virus infections and arteriosclerosis.

In a sixth embodiment of the fourth aspect, which is also an embodimentof the fifth embodiment, the tumour disease is selected from the groupcomprising mesenchymal tumours, epithelial tumours, benign tumours,malignant tumours and metastasising tumours.

In a seventh embodiment of the fourth aspect, which is also anembodiment of the third, fourth, fifth and sixth embodiment, the targetmolecule is HMGA and the disease is selected from the group comprisingcarcinomas of the prostate, pancreas, thyroid, cervix, stomach, breast,colon/rectum, ovaries; neuroblastomas; lymphomas, uterine leiomyomas;lipomas; endometrial polyps; chondroid hamartomas of the lungs;pleomorphic adenomas of the salivary glands; haemangiopericytomas;chondromatous tumours; aggressive angiomyxomas; diffuse astrocytomas;osteoclastomas; skin cancer; Burkitt's lymphoma; Lewis lung cancer;leukaemia; non-small-cell lung cancer; as well as in each casemetastases and/or metastasising forms thereof.

In an eighth embodiment of the fourth aspect, which is also anembodiment of the fifth embodiment, the arteriosclerosis is triggered byformation of arteriosclerotic plaques mediated by HMGA1, HMGA1a, HMG1band/or HMGA2.

In a ninth embodiment of the fourth aspect, which is also an embodimentof the first, second, third, fourth, fifth, sixth and seventhembodiment, the intracellular target molecule is presentintracellularly.

According to a fifth aspect of the invention the object is achieved by acomposition comprising a L-nucleic acid binding to an intracellulartarget molecule, and a delivery vehicle.

In a first embodiment of the fifth aspect the delivery vehicle is adelivery vehicle suitable for the intracellular delivery of theL-nucleic acid.

In a second embodiment of the fifth aspect, which is also an embodimentof the first embodiment, the delivery vehicle is selected from the groupcomprising vehicles, conjugates and physical means.

In a third embodiment of the fifth aspect, which is also an embodimentof the second embodiment, the delivery vehicle is a vehicle selectedfrom the group comprising liposomes, nanoparticles, microparticles,cyclodextrins or dendrimers, or a vesicle consisting of polypeptides,polyethyleneimine and/or amphipathic molecules.

In a fourth embodiment of the fifth aspect, which is also an embodimentof the second embodiment, the delivery vehicle is a conjugate, whereinthe conjugate is a conjugate for the receptor-mediated endocytosis, aconjugate with a fusogenic peptide, a conjugate with a signal peptide, aconjugate with a nucleic acid, preferably a conjugate with a spiegelmer,or a lipophilic conjugate.

In a fifth embodiment of the fifth aspect, which is also an embodimentof the second embodiment, the delivery vehicle is a physical means, thephysical means preferably being selected from the group comprisingelectroporation, iontophoresis, pressure, ultrasound and shock waves.

In a sixth embodiment of the fifth aspect, which is also an embodimentof the third embodiment, the delivery vehicle comprisespolyethyleneimine.

In a seventh embodiment of the fifth aspect, which is also an embodimentof the sixth embodiment, the polyethyleneimine is a branchedpolyethyleneimine with a molecular weight of about 25 kDa.

In an eighth embodiment of the fifth aspect, which is also an embodimentof the sixth and seventh embodiment, the polyethyleneimine forms amicelle or a micelle-like structure.

In a ninth embodiment of the fifth aspect, which is also an embodimentof the first, second, third, fourth, fifth, sixth, seventh and eighthembodiment, the L-nucleic acid is a spiegelmer.

In a tenth embodiment of the fifth aspect, which is also an embodimentof the ninth embodiment, the spiegelmer carries a modification, the saidmodification being selected from the group comprising PEG residues.

In an eleventh embodiment of the fifth aspect, which is also anembodiment of the tenth embodiment, the PEG residue has a molecularweight of about 1,000 to 10,000 Da, preferably a molecular weight ofabout 1,500 to 2,500 Da and most preferably a molecular weight of about2,000 Da.

In a twelfth embodiment of the fifth aspect, which is also an embodimentof the tenth and eleventh embodiment, the modification is bound to the5′ terminus or to the 3′ terminus of the L-nucleic acid.

In a thirteenth embodiment of the fifth aspect, which is also anembodiment of the ninth, tenth, eleventh and twelfth embodiment, in thecomposition the ratio of the total number of nitrogen groups of thepolyethyleneimine to the total number of phosphate groups of the nucleicacid contained in the composition is about 1 to 20, preferably about 1.5to 10, more preferably about 2 to 5 and most preferably about 2 to 3.

In a fourteenth embodiment of the fifth aspect, which is also anembodiment of the first, second, third, fourth, fifth, sixth, seventh,eighth, ninth, tenth, eleventh, twelfth and thirteenth embodiment, thecomposition provides the L-nucleic acid intracellularly.

According to a sixth aspect of the invention the object is achieved bythe pharmaceutical composition comprising a composition according to thefifth aspect, and a pharmaceutically acceptable carrier.

In an embodiment of the use according to the first aspect the L-nucleicacid is a composition according to the fifth aspect.

In an embodiment of the method according to the second aspect theL-nucleic acid is a composition according to the fifth aspect.

In an embodiment of the use according to the third aspect the L-nucleicacid is a composition according to the fifth aspect.

In an embodiment of the use according to the fourth aspect the L-nucleicacid is a composition according to the fifth aspect.

According to a seventh aspect of the invention the object is achieved byan HMGA-binding nucleic acid, characterised in that the nucleic acidcomprises a section Box A1 and a section Box A2, wherein the section BoxA1 and the section Box A2 are joined to one another by an intermediatesection and wherein Box A1 and Box A2 are selected individually andindependently of one another from the group comprising GGGCG, GGGUG andGGGAG.

In a first embodiment of the seventh aspect the intermediate sectionconsists either of an intermediate section Z1 comprising six or sevennucleotides, or of an intermediate section Z2 comprising 12 to 25nucleotides.

In a second embodiment of the seventh aspect, which is also anembodiment of the first embodiment, the nucleic acid at the 5′ end ofthe section Box A1 has a first section and at the 3′ end of the sectionBox A2 has a second section, wherein preferably both sectionsindependently of one another comprise four to eight nucleotides.

In a third embodiment of the seventh aspect, which is also an embodimentof the second embodiment, the two sections are at least partly orcompletely hybridised with one another, the hybridisation extending overfour to eight nucleotide pairs.

In a fourth embodiment of the seventh aspect, which is also anembodiment of the second and third embodiments, the nucleic acid has atthe 5′ end of the section Box A1 a section Helix A1 and at the 3′ end ofthe section Box A2 a section Helix A2, wherein preferably the sectionHelix A1 comprises four to eight nucleotides and preferably the sectionHelix A2 comprises four to eight nucleotides, and wherein preferably thesection Helix A1 forms the first section at the 5′ end of the sectionBox A1 or a part thereof, and wherein preferably the section Helix A2forms the second section at the 3′ end of the section Box A2 or a partthereof, the length of the section Helix A1 being independent of thelength of the section Helix A2.

In a fifth embodiment of the seventh aspect, which is also an embodimentof the fourth embodiment, the sections Helix A1 and Helix A2 are atleast partly or completely hybridised with one another, thehybridisation extending over four to eight nucleotide pairs.

In a sixth embodiment of the seventh aspect, which is also an embodimentof the fourth and fifth embodiment, between the 3′ end of the sectionHelix A1 and the 5′ end of the section Box A1 a section Helix B1 isarranged, and between the 3′ end of the section Box A2 and the 5′ end ofthe section Helix A2 a section Helix B2 is arranged, wherein preferablythe length of the section Helix B1 and Helix B2 comprises in each caseindividually and independently a length of four to eight nucleotides.

In a seventh embodiment of the seventh aspect, which is also anembodiment of the sixth embodiment, the sections Helix B1 and Helix B2are at least partly or completely hybridised with one another, thehybridisation extending over four to eight nucleotide pairs.

In an eighth embodiment of the seventh aspect, which is also anembodiment of the sixth and seventh embodiment, zero to five nucleotidesare arranged between the 3′ end of the section Helix A1 and the 5′ endof the section Helix B1.

In a ninth embodiment of the seventh aspect, which is also an embodimentof the eighth embodiment, two nucleotides are arranged between the 3′end of the section Helix A1 and the 5′ end of the section Helix B1.

In a tenth embodiment of the seventh aspect, which is also an embodimentof the sixth, seventh, eighth and ninth embodiment, zero to sixnucleotides are arranged between the 3′ end of the section Helix B2 andthe 5′ end of the section Helix A2.

In an eleventh embodiment of the seventh aspect, which is also anembodiment of the tenth embodiment, preferably insofar as this is anembodiment of the ninth embodiment, a nucleotide is arranged between the3′ end of the section Helix B2 and the 5′ end of the section Helix A2.

In a twelfth embodiment of the seventh aspect, which is also anembodiment of the sixth, seventh, eighth, ninth, tenth and eleventhembodiment, the sum of the nucleotides of section Helix A1 and ofsection Helix B1 is ten to twelve nucleotides, and the sum of thenucleotides of section Helix A2 and of section Helix B2 is ten to twelvenucleotides.

In a thirteenth embodiment of the seventh aspect, which is also anembodiment of the twelfth embodiment, the sum of the hybridisednucleotides from the hybridisation of section Helix A1 with sectionHelix A2 and of section Helix B1 with section Helix B2 is ten to twelvenucleotide pairs.

In a fourteenth embodiment of the seventh aspect, which is also anembodiment of the sixth, seventh, eighth, ninth, tenth, eleventh,twelfth and thirteenth embodiment, preferably of the sixth or seventhembodiment, the nucleic acid does not comprise a section Helix A1 andHelix A2, whereby the section Helix B1 is arranged at the 5′ end of thenucleic acid and the Helix B2 is arranged at the 3′ end, whereinpreferably the length of the section Helix B1 and Helix B2 comprises ineach case individually and independently a length of four to eightnucleotides.

In a fifteenth embodiment of the seventh aspect, which is also anembodiment of the fourteenth embodiment, the sections Helix B1 and HelixB2 are at least partly or completely hybridised with one another, thehybridisation extending over four to eight nucleotide pairs.

In a sixteenth embodiment of the seventh aspect, which is an embodimentof the fourth and fifth embodiment, one to five nucleotides are arrangedbetween the 3′ end of the section Helix A1 and the 5′ end of the sectionBox A1, and one to three nucleotides are arranged between the 3′ end ofthe section Box A2 and the 5′ end of the section Helix A2.

In a seventeenth embodiment of the seventh aspect, which is also anembodiment of the sixth, seventh, eighth, ninth, tenth, eleventh,twelfth, thirteenth, fourteenth and fifteenth embodiment, twonucleotides are arranged between the 3′ end of the section Helix B1 andthe 5′ end of the section Box A1, and one to seven nucleotides arearranged between the 3′ end of the section Box A2 and the 5′ end of thesection Helix B2.

In an eighteenth embodiment of the seventh aspect, which is also anembodiment of the first, second, third, fourth, fifth, sixth, seventh,eighth and tenth embodiment, insofar as this is an embodiment of thesixth, seventh and eighth embodiment, of the twelfth and thirteenthembodiment, insofar as these are embodiments of the sixth, seventh,eighth and tenth embodiments, of the fourteenth and fifteenthembodiment, insofar as these are embodiments of the sixth, seventh,eighth, tenth, twelfth and thirteenth embodiment, or of the seventeenthembodiment, insofar as these are embodiments of the sixth, eighth,tenth, twelfth, thirteenth and fifteenth embodiment, in each case in theherein restricted scope, the intermediate section Z1 comprises six orseven nucleotides.

In a nineteenth embodiment of the seventh aspect, which is also anembodiment of the eighteenth embodiment, the intermediate section Z1comprises the sequence N₁N₂GN₈N₃N₄N₅, wherein

N₁=U, C, A or G; N₂=G or U; N₃=U or C; N₄=U or A; N₅=G or A; and

N₈=U or is absent.

In a twentieth embodiment of the seventh aspect, which is also anembodiment of the nineteenth embodiment, the nucleic acid comprises asection Box A1 and a section Box A2, wherein the 3′ end of the sectionBox A1 is joined directly to the 5′ end of the intermediate section Z1,and the 3′ end of the intermediate section Z1 is joined directly to the5′ end of the section Box A2.

In a twenty-first embodiment of the seventh aspect, which is also anembodiment of the eighteenth, nineteenth and twentieth embodiment, inparticular of the twentieth embodiment, the nucleic acid comprises asection Helix B1 and a section Helix B2.

In a twenty-second embodiment of the seventh aspect, which is also anembodiment of the twenty-first embodiment, the sections Helix B1 andHelix B2 comprise in each case individually and independently of oneanother four to eight nucleotides, which are preferably completely orpartly hybridised with one another.

In a twenty-third embodiment of the seventh aspect, which is also anembodiment of the twenty-first and twenty-second embodiment, twonucleotides N₆, N₇ are arranged between the 3′ end of the section HelixB1 and the 5′ end of the section Box A1 in the 5′ 3′ direction, whereinN₆ is G, A or U, and N₇ is G or U.

In a twenty-fourth embodiment of the seventh aspect, which is also anembodiment of the twenty-first, twenty-second and twenty-thirdembodiment, there is no nucleotide between the 3′ end of the section BoxA2 and the 5′ end of the section Helix B2, or the nucleotide sequenceGN_(y) is arranged in the 5′ 3′ direction, wherein N_(y) comprises zeroto six nucleotides, preferably 0 or 6 nucleotides.

In a twenty-fifth embodiment of the seventh aspect, which is also anembodiment of the eighteenth, nineteenth, twentieth, twenty-first,twenty-second, twenty-third and twenty-fourth embodiment, the nucleicacid comprises a section Helix A1 and Helix A2.

In a twenty-sixth embodiment of the seventh aspect, which is also anembodiment of the twenty-fifth embodiment, the sections Helix A1 andHelix A2 comprise in each case individually and independently of oneanother four to eight nucleotides, wherein preferably the sections HelixA1 and Helix A2 are completely or partly hybridised with one another.

In a twenty-seventh embodiment of the seventh aspect, which is also anembodiment of the twenty-fifth and twenty-sixth embodiment, anucleotides sequence N_(X) is arranged between the 3′ end of the sectionHelix A1 and the 5′ end of the section Helix B1, wherein N_(X) compriseszero to five nucleotides.

In a twenty-eighth embodiment of the seventh aspect, which is also anembodiment of the twenty-fifth, twenty-sixth and twenty-seventhembodiment, a nucleotide sequence N_(z) is arranged between the 3′ endof the section Helix B2 and the 5′ end of the section Helix A2, whereinN_(z) comprises zero to six nucleotides.

In a twenty-ninth embodiment of the seventh aspect, which is also anembodiment of the twenty-first, twenty-second, twenty-third,twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh andtwenty-eighth embodiment, the sum of the hybridised nucleotides from thehybridisation of section Helix A1 with section Helix A2 and of sectionHelix B1 with section Helix B2 is ten to twelve nucleotide pairs.

In a thirtieth embodiment of the seventh aspect, which is also anembodiment of the twenty-fourth, twenty-fifth, twenty-sixth,twenty-seventh, twenty-eighth and twenty-ninth embodiment, thenucleotide sequence GN_(y) is arranged between the 3′ end of the sectionBox A2 and the 5′ end of the section Helix B2 in the 5′-3′ direction,wherein N_(y) comprises zero to six nucleotides, preferably 0 or 6nucleotides.

In a thirty-first embodiment of the seventh aspect, which is also anembodiment of the thirtieth embodiment, the HMGA-binding nucleic acidcomprises the following structure

wherein

N₁=U, C, A or G; N₂=G or U; N₃=U or C; N₄=U or A; N₅=G or A; N₆=G, A orU; N₇=G or U;

N₈=U or is no nucleotide;N_(x)=zero to five nucleotides;N_(y)=zero or six nucleotides; andN_(z)=zero to six nucleotides;the section Box A1 and section Box A2 are selected in each caseindividually and independently of one another from the group ofnucleotide sequences comprising GGGCG, GGGUG and GGGAG;the section Helix A1 and the section Helix A2 comprise in each caseindividually and independently of one another four to eight nucleotides,wherein preferably the sections Helix A1 and Helix A2 are completely orpartly hybridised with one another, andthe sections Helix B1 and Helix B2 comprise in each case individuallyand independently of one another four to eight nucleotides, whereinpreferably the sections Helix B1 and Helix B2 are completely or partlyhybridised with one another and the hybridising region comprises four toeight nucleotides, and wherein the sum of the hybridised nucleotidesfrom the hybridisation of section Helix A1 with section Helix A2 and ofsection Helix B1 with section Helix B2 is 10 to 12 nucleotide pairs.

In a thirty-second embodiment of the seventh aspect, which is also anembodiment of the thirtieth and thirty-first embodiment, theHMGA-binding nucleic acid comprises a sequence selected from the groupcomprising SEQ. ID. No. 1, SEQ. ID. No. 2, SEQ. ID. No. 3, SEQ. ID. No.5, SEQ. ID. No. 6, SEQ. ID. No. 7 and SEQ. ID. No. 13.

In a thirty-third embodiment of the seventh aspect, which is also anembodiment of the twenty-fourth, twenty-fifth, twenty-sixth,twenty-seventh, twenty-eighth and twenty-ninth embodiment, the 3′ end ofthe section Box A2 is joined directly to the 5′ end of the section HelixB2.

In a thirty-fourth embodiment of the seventh aspect, which is also anembodiment of the thirty-third embodiment, the HMGA-binding nucleic acidhas the following structure

wherein

N₁=U, C, A or G; N₂=G or U; N₃=U or C; N₄=U or A; N₅=G or A; N₆=G, A orU; N₇=G or U;

N₈=U or is no nucleotide;N_(x)=zero to five nucleotides; andN_(z)=zero to six nucleotides;the section Box A1 and Section Box A2 are selected in each caseindividually and independently of one another from the group ofnucleotide sequences comprising GGGCG, GGGUG and GGGAG;the section Helix A1 and the section Helix A2 comprise in each caseindividually and independently of one another four to eight nucleotides,wherein preferably the sections Helix A1 and Helix A2 are completely orpartly hybridised with one another, andthe sections Helix B1 and Helix B2 comprise in each case individuallyand independently of one another four to eight nucleotides, whereinpreferably the sections Helix B1 and Helix B2 are completely or partlyhybridised with one another and the hybridising region comprises four toeight nucleotides, and wherein the sum of the hybridised nucleotidesfrom the hybridisation of section Helix A1 with section Helix A2 and ofsection Helix B1 with section Helix B2 is 10 to 12 nucleotide pairs.

In a thirty-fifth embodiment of the seventh aspect, which is also anembodiment of the thirty-third and thirty-fourth embodiment, theHMGA-binding nucleic acid comprises a sequence including SEQ. ID. No. 3.

In a thirty-sixth embodiment of the seventh aspect, which is also anembodiment of the thirty-first embodiment, the HMGA-binding nucleic acidcomprises the following structure

In a thirty-seventh embodiment of the seventh aspect, which is also anembodiment of the thirty-fourth embodiment, the HMGA-binding nucleicacid comprises the following structure

In a thirty-eighth embodiment of the seventh aspect, which is also anembodiment of the thirty-sixth embodiment, the HMGA-binding nucleic acidcomprises a sequence which is selected from the group including SEQ. ID.No. 15 and SEQ. ID. No. 16.

In a thirty-ninth embodiment of the seventh aspect, which is also anembodiment of the first, second, third, fourth, fifth, sixth, seventh,eighth, ninth, tenth, eleventh, twelfth, thirteenth and sixteenth orseventeenth embodiment of the seventh aspect, the HMGA-binding nucleicacid comprises an intermediate section Z₂ which comprises 12 to 25nucleotides.

In a fortieth embodiment of the seventh aspect, which is also anembodiment of the thirty-ninth embodiment, the HMGA-binding nucleic acidcomprises an intermediate section Z2, a section Helix C1 and a sectionHelix C2.

In a forty-first embodiment of the seventh aspect, which is also anembodiment of the fortieth embodiment, a central section N_(c) isarranged between the section Helix C1 and the section Helix C2 of theHMGA-binding nucleic acid.

In a forty-second embodiment of the seventh aspect, which is also anembodiment of the fortieth or forty-first embodiment, the length of thesection Helix C1 and Helix C2 of the HMGA-binding nucleic acid areidentical.

In a forty-third embodiment of the seventh aspect, which is also anembodiment of the fortieth, forty-first and forty-second embodiment, thelength of the section Helix C1 and Helix C2 of the HMGA-binding nucleicacid is individually and independently three to six nucleotides.

In a forty-fourth embodiment of the seventh aspect, which is also anembodiment of the fortieth, forty-first, forty-second and forty-thirdembodiment, the sections Helix C1 and Helix C2 of the HMGA-bindingnucleic acid are completely or party hybridised with one another.

In a forty-fifth embodiment of the seventh aspect, which is also anembodiment of the thirty-ninth, fortieth, forty-first, forty-second,forty-third and forty-fourth embodiment, the central section N_(c) ofthe HMGA-binding nucleic acid comprises three to five nucleotides.

In a forty-sixth embodiment of the seventh aspect, which is also anembodiment of the thirty-ninth, fortieth, forty-first, forty-second,forty-third, forty-fourth and forty-fifth embodiment, the HMGA-bindingnucleic acid comprises a section Box A1 and a section Box A2, wherein anucleotide sequence N_(b) is arranged between the 3′ end of the sectionBox A1 and the 5′ end of the section Helix C1 and comprises threenucleotides.

In a forty-seventh embodiment of the seventh aspect, which is also anembodiment of the thirty-ninth, fortieth, forty-first, forty-second,forty-third, forty-fourth, forty-fifth and forty-sixth embodiment, theHMGA-binding nucleic acid comprises a section Box A1 and a section BoxA2, wherein a nucleotide sequence N_(d) is arranged between the 3′ endof the section Helix C2 and the 5′ end of the section Box A2 andcomprises two to five nucleotides.

In a forty-eighth embodiment of the seventh aspect, which is also anembodiment of the thirty-ninth, fortieth, forty-first, forty-second,forty-third, forty-fourth, forty-fifth, forty-sixth and forty-seventhembodiment, the HMGA-binding nucleic acid comprises a section Helix A1and a section Helix A2.

In a forty-ninth embodiment of the seventh aspect, which is also anembodiment of the forty-eighth embodiment, the sections Helix A1 andHelix A2 of the HMGA-binding nucleic acid comprise in each caseindividually and independently of one another five to six nucleotides,wherein preferably the section Helix A1 and the section Helix A2 arecompletely or partly hybridised with one another.

In a fiftieth embodiment of the seventh aspect, which is also anembodiment of the forty-eighth and forty-ninth embodiment, a nucleotidesequence N_(a) is arranged between the 3′ end of the section Helix A1and the 5′ end of the section Box A1 of the HMGA-binding nucleic acid,wherein N_(a) comprises one to five nucleotides.

In a fifty-first embodiment of the seventh aspect, which is also anembodiment of the forty-eighth, forty-ninth and fiftieth embodiment, anucleotide sequence GN_(e) is arranged between the 3′ end of the sectionBox A2 and the 5′ end of the section Helix A2 of the HMGA-bindingnucleic acid in the 5′-3′ direction, wherein, N_(e) comprises one to twonucleotides, preferably A or UU.

In a fifty-second embodiment of the seventh aspect, which is also anembodiment of the forty-eighth, forty-ninth, fiftieth and fifty-firstembodiment, the section Helix C1 and the section Helix C2 of theHMGA-binding nucleic acid have in each case individually andindependently of one another a length of five or six nucleotides,wherein preferably the sections Helix C1 and Helix C2 are completely orpartly hybridised with one another.

In a fifty-third embodiment of the seventh aspect, which is also anembodiment of the fifty-second embodiment, the HMGA-binding nucleic acidhas the following structure:

whereinN_(a)=one to five nucleotides;N_(b)=three nucleotides;N_(c)=three to five nucleotides;N_(d)=two to five nucleotides; andN_(e)=one to two nucleotides, preferably A or UU;the section Box A1 and the section Box A2 are selected in each caseindividually and independently of one another from the group comprisingGGGCG, GGGUG and GGGAG,the sections Helix A1 and Helix A2 comprise in each case individuallyand independently of one another five or six nucleotides, andthe sections Helix C1 and Helix C2 comprise in each case five or sixnucleotides, which are preferably completely or partly hybridised withone another.

In a fifty-fourth embodiment of the seventh aspect, which is also anembodiment of the fifty-third embodiment, the HMGA-binding nucleic acidcomprises a sequence which is selected from the group comprising SEQ.ID. No. 8, SEQ. ID. No. 9, SEQ. ID. No. 10, SEQ. ID. No. 11, SEQ. ID.No. 14, SEQ. ID. No. 22 and SEQ. ID. No. 24.

In a fifty-fifth embodiment of the seventh aspect, which is also anembodiment of the thirty-ninth, fortieth, forty-first, forty-second,forty-third and forty-fourth embodiment, the nucleic acid comprises asection Box 1 and a section Helix C1 of the HMGA-binding nucleic acid,wherein a nucleotide A is arranged between the 3′ end of the section BoxA1 and the 5′ end of the section Helix C1.

In a fifty-sixth embodiment of the seventh aspect, which is also anembodiment of the fifty-fifth embodiment, the HMGA-binding nucleic acidcomprises a section Helix C2 and a section Box A2, wherein a nucleotideG is arranged between the 3′ end of the section Helix C2 and the 5′ endof the section Box A2.

In a fifty-seventh embodiment of the seventh aspect, which is also anembodiment of the fifty-fifth or fifty-sixth embodiment, the centralsection N_(c) of the HMGA-binding nucleic acid comprises fournucleotides, wherein N_(c) is preferably GAUG.

In a fifty-eighth embodiment of the seventh aspect, which is also anembodiment of the fifty-fifth, fifty-sixth and fifty-seventh embodiment,the HMGA-binding nucleic acid comprises a section Helix B1 and a sectionHelix B2.

In a fifty-ninth embodiment of the seventh aspect, which is also anembodiment of the fifty-eighth embodiment, the sections Helix B1 andHelix B2 of the HMGA-binding nucleic acid comprise individually andindependently of one another in each case five nucleotides, whereinpreferably the section Helix B1 is hybridised with the section Helix B2.

In a sixtieth embodiment of the seventh aspect, which is also anembodiment of the fifty-eighth or fifty-ninth embodiment, a nucleotidesequence comprising two nucleotides N_(j) is arranged between the 3′ endof the section Helix B1 and the 5′ end of the section Box A1 of theHMGA-binding nucleic acid, wherein N_(j) is preferably AG.

In a sixty-first embodiment of the seventh aspect, which is also anembodiment of the fifty-eighth, fifty-ninth and sixtieth embodiment, anucleotide G is arranged between the 3′ end of the section Box A2 andthe 5′ end of Helix B2 of the HGMA-binding nucleic acid.

In a sixty-second embodiment of the seventh aspect, which is also anembodiment of the fifty-fifth, fifty-sixth, fifty-seventh, fifty-eighth,fifty-ninth, sixtieth and sixty-first embodiment, the HMGA-bindingnucleic acid comprises a section Helix A1 and a section Helix A2.

In a sixty-third embodiment of the seventh aspect, which is also anembodiment of the sixty-second embodiment, the sections Helix A1 andHelix A2 of the HMGA-binding nucleic acid comprise individually andindependently of one another in each case six nucleotides and preferablythe section Helix A1 and the section Helix A2 are hybridised with oneanother.

In a sixty-fourth embodiment of the seventh aspect, which is also anembodiment of the sixty-second and sixty-third embodiment, a nucleotidesequence comprising two nucleotides N_(i) is arranged between the 3′ endof the section Helix A1 and the 5′ end of the section Helix B1, whereinN_(i) is preferably CA.

In a sixty-fifth embodiment of the seventh aspect, which is also anembodiment of the sixty-second, sixty-third and sixty-fourth embodiment,a nucleotide A is arranged between the 3′ end of the section Helix B2and the 5′ end of the section Helix A2.

In a sixty-sixth embodiment of the seventh aspect, which is also anembodiment of the fifty-fifth to sixty-fifth embodiment, the sectionsHelix C1 and Helix C2 comprise in each case three nucleotides, whereinpreferably the section Helix C1 and Helix C2 are hybridised with oneanother.

In a sixty-seventh embodiment of the seventh aspect, which is also anembodiment of the sixty-sixth embodiment, the HMGA-binding nucleic acidhas the following structure:

whereinN_(i)=two nucleotides, preferably CA;N_(j)=two nucleotides, preferably AG;N_(c)=four nucleotides, preferably GAUG;the sections Box A1 and Box A2 are in each case selected individuallyand independently of one another from the group comprising the sequencesGGGCG, GGGUG and GGGAG;the sections Helix A1 and Helix A2 comprise in each case individuallyand independently six nucleotides, which are preferably hybridised withone another;the sections Helix B1 and Helix B2 comprise in each case individuallyand independently five nucleotides, wherein preferably the section HelixB1 and the section Helix B2 are hybridised with one another, andthe section Helix C1 and Helix C2 comprise in each case individually andindependently three nucleotides, wherein preferably the sections HelixC1 and Helix C2 are hybridised with one another.

In a sixty-eighth embodiment of the seventh aspect, which is also anembodiment of the sixty-seventh embodiment, the HMGA-binding nucleicacid comprises a sequence which is selected from the group includingSEQ. ID. No. 12.

In a sixty-ninth embodiment of the seventh aspect, which is also anembodiment of the second to sixty-seventh embodiment, the nucleic acidis one that binds to transcription factors, in particular transcriptionfactors comprising an AT hook.

According to the invention the object is achieved in an eighth aspect bya nucleic acid, which binds to a transcription factor comprising an AThook, wherein the nucleic acid has a structure according to the seventhaspect.

In an embodiment of the composition according to the sixth aspect theL-nucleic acid is a nucleic acid according to the seventh aspect.

In an embodiment of the use according to the first aspect the L-nucleicacid is a nucleic acid according to the seventh aspect.

In an embodiment of the method according to the second aspect theL-nucleic acid is a nucleic acid according to the seventh aspect.

In an embodiment of the use according to the third aspect the L-nucleicacid is a nucleic acid according to the seventh aspect.

In an embodiment of the method according to the fourth aspect theL-nucleic acid is a nucleic acid according to the seventh aspect.

According to the invention the object is achieved in a ninth aspect by amethod for screening an HMGA antagonist or HMGA agonist, comprising thefollowing steps:

-   -   providing a candidate HMGA antagonist and/or a candidate HMGA        agonist,    -   providing a nucleic acid according to the seventh aspect,    -   providing a test system which delivers a signal in the presence        of an HMGA antagonist and/or an HMGA agonist, and    -   determining whether the candidate HMGA antagonist is an HMGA        antagonist and/or whether the candidate HMGA agonist is an HMGA        agonist.

According to the invention the object is achieved in a tenth aspect by amethod for screening an HMGA agonist and/or an HMGA antagonist,comprising the following steps:

-   -   providing an HMGA immobilised on a phase, preferably a solid        phase,    -   providing a nucleic acid according to the seventh aspect,        preferably a nucleic acid according to the seventh aspect which        is labelled,    -   adding a candidate HMGA agonist and/or a candidate HMGA        antagonist, and    -   determining whether the candidate HMGA agonist is an HMGA        agonist and/or whether the candidate HMGA antagonist is an HMGA        antagonist.

In an embodiment of the tenth aspect it is envisaged that thedetermination is carried out by testing whether the nucleic acid isreplaced by the candidate HMGA agonist or by the candidate HMGAantagonist.

According to the invention the object is achieved in an eleventh aspectby a kit for the detection of HMGA, comprising a nucleic acid accordingto the seventh aspect.

According to the invention the object is achieved in a twelfth aspect byan HMGA antagonist which is obtainable by a method according to thetenth aspect.

According to the invention the object is achieved in a thirteenth aspectby an HMGA agonist which is obtainable by a method according to thetenth aspect.

According to the invention the object is achieved in a fourteenth aspectby a complex comprising an HMGA protein and a nucleic acid according tothe seventh aspect.

The present invention is based on the surprising result that, contraryto the received opinion in the prior art, it is possible to useL-nucleic acids and in particular spiegelmers in order to addressintracellular target molecules. The intracellular target molecules arepreferably target molecules which are present in a cell. The propertiesinherent in the functional L-nucleic acids due to their structure ofL-nucleotides, such as high specificity of the interaction with theirtarget molecules with at the same time a high stability and absence oftoxic or immunologically active decomposition products when theL-nucleic acids are used in biological systems and in particular in theanimal and human body, does not however allow the cellular mechanisms tobe utilised in order, as in the case of intramers, for L-nucleic acidsto be coded by a plasmid or generally a vector and thus provide theactually functional nucleic acid by the intracellularly occurringprocess of transcription.

This inescapable dilemma is solved by the present invention: functionalL-nucleic acids and in particular spiegelmers can be transported througha cytoplasmic membrane while retaining their specificity as regardstheir binding to their target molecule, and their activity. Thispermeability of the functional L-nucleic acids is inherent inspiegelmers and can be enhanced still further by the use of deliveryvehicles or delivery techniques. Without hereinafter wishing to bespecific in this matter, the present inventors start from the assumptionthat functional L-nucleic acids can per se overcome the cytoplasmicmembrane, and with the participation of endosomal transport mechanismsin overcoming the cytoplasmic membrane, are able to free this from thevesicle structures that are thereby formed, with the adoption of atwo-dimensional or three-dimensional structure, which allows thespecific interaction of the functional nucleic acid with its targetmolecule. With the technical teaching disclosed herein, the principledeveloped for aptamers of utilising intracellular transcriptionmechanisms in order to create aptamers in the cell is intentionallyavoided, and for the first time means are provided for using functionalL-nucleic acids and in particular spiegelmers in cells.

As employed herein in a preferred embodiment, the term functionalnucleic acids denotes those nucleic acids that are different fromstructural, in particular naturally occurring structural nucleic acidssuch as rRNAs or that are different from coding nucleic acids such asmRNAs. In particular functional nucleic acids are nucleic acids which,on account of their two-dimensional and/or three-dimensional structure,are able to bind to target molecules. In a particularly preferredembodiment the binding to the target molecule takes place not byhybridisation or base pairing on the basis of Watson-Crick base pairingsor a Hoogsteen base pairing. Particularly preferred functional nucleicacids are aptamers and spiegelmers.

A L-nucleic acid is in a preferred embodiment a nucleic acid that iscompletely, substantially or partly synthesised from L-nucleotides. Itis particularly preferred if the L-nucleic acid consists completely ofL-nucleotides. The term “substantially” denotes in this connection anembodiment in which that part of the L-nucleic acid which is responsiblefor the interaction with the target molecule, or that part whichmediates the binding to the target molecule, consist of L-nucleotides oris synthesised from these.

As used herein, a functional L-nucleic acid is a functional nucleic acidwhich is completely, substantially or partly synthesised fromL-nucleotides.

The synthesis of L-nucleic acids is known to the person skilled in theart in this field and is described for example in Nolte et al., Nat.Biotech, 14, 1116-1119, 1996.; and Klussmann et al., Nat. Biotechnol,14, 1112-1115, 1996.

The basic process for the production of aptamers is described forexample in Tuerck et al. Science, 248, 505-510, 1990; or Ellington etal. Nature, 346, 818-822, 1990, while the basic process for theproduction of spiegelmers is described for example in Nolte et al., Nat.Biotech, 14, 1116-1119, 1996.; or Klussmann et al., Nat. Biotechnol, 14,1112-1115, 1996. Spiegelmers are thus aptamers which consist ofL-nucleotides instead of D-nucleotides. In connection with theproduction of aptamers and spiegelmers, the term target molecule denotesthat molecule which is used in the selection process to produce aptamersand spiegelmers, or denotes that molecule which is ultimately bound bythe aptamer or the spiegelmer.

In a preferred embodiment an intracellularly active agent is a chemicalcompound which when present in a cell is able to bind to a molecule. Inthis connection it is particularly preferred if the cell is a cell thatexists isolated in a tissue or an organ, but preferably not in a humanor animal body. If the intracellularly active agent is a spiegelmer,then preferably it is an intracellularly active agent if it is able tobind to an intracellular target molecule. Alternatively the spiegelmeris an intracellularly active agent if it is able to bind to its targetmolecule under conditions such as exist in a cell. Tests in order todetermine these properties are known to the person skilled in the art inthis field, and include for example equilibrium binding assays underbuffer conditions such as exist intracellularly (ionic strength andsolute composition, pH, temperature), as disclosed in Example 1.

In a preferred embodiment the target molecule of the L-nucleic acid, inparticular of the functional L-nucleic acid, is an intracellularreceptor. An intracellular receptor, as used herein, is preferably achemical compound or a chemical structure or respectively a partthereof, with which the functional L-nucleic acid interacts, and ispreferably a compound or structure to which the functional L-nucleicacid binds, wherein the intracellular receptor, i.e. the chemicalcompound or the chemical structure or respectively a part thereof, ispresent intracellularly, i.e. is present in a cell, as is preferablydescribed in the preceding paragraph. In this connection it is possiblewithin the scope of the present invention for the intracellular receptorto be the target molecule in the creation of the functional nucleicacid, in particular the functional L-nucleic acid.

In one embodiment the term “receptor” denotes any interaction partner,preferably a specifically binding interaction partner of the functionalnucleic acid, i.e. denotes an interaction partner interacting with thefunctional nucleic acid, which has a specific spatial structure, chargedistribution, hydrophobicity distribution, etc. In a particularlypreferred embodiment the interaction partner corresponds to the targetmolecule of the functional nucleic acid, as was used in the creation ofthe functional nucleic acid. In this connection it is within the scopeof the present invention that a receptor can also be different from thetarget molecule used in the creation of the functional nucleic acid,though the specific interaction is due to a cross reactivity of thefunctional nucleic acid between the interaction partner and the targetmolecule used in the creation of the functional nucleic acid.

In a preferred embodiment the term “intracellular receptor” denotes areceptor that is present in a cell, or a receptor that can be present ina cell, that occurs under natural circumstances in a cell, or that undersuch circumstances exist in a cell. In this connection it isparticularly preferred if the cell is a cell that occurs isolated in atissue or an organ, but preferably not in a human or animal body. Asused herein, the term “intracellular receptor” however also denotes areceptor that is present under conditions such as exist in a cell.

In a preferred embodiment the term “cell” denotes a cell which isselected from the group comprising prokaryotic and eukaryotic cells.Preferably the eukaryotic cell is selected from the group comprisingfungal cells, plant cells, animal cells and human cells. In analternative embodiment the term cell generally denotes herein acompartment bounded by a phospholipid double membrane, which in apreferred embodiment corresponds to a cytoplasmic membrane, and which isseparated by the membrane from the surroundings. The separation from thesurroundings is in this connection not a complete separation, but allowsan energy transfer and a mass transfer (substance exchange) between thecell and the surroundings. The mass transfer is preferably restricted.In the case where the cell is separated from the surroundings by acytoplasmic membrane or by a membrane similar to a cytoplasmic membrane,the restriction of the mass transfer is defined by the transportproperties of the membrane. In one embodiment the term cell herein thusalso includes vesicles and/or compartments of a prokaryotic oreukaryotic cell as defined herein, which in turn are present or may bepresent both within a prokaryotic or eukaryotic cell, as well as outsidesuch a prokaryotic or eukaryotic cell, for example as vesicles or partsof a prokaryotic or eukaryotic cell surrounded by a cytoplasmicmembrane, which in one embodiment can be present in a body fluid. In apreferred embodiment, in a cell according to the second alternativeembodiment it is envisaged that the conditions within such a cellcorrespond substantially to those occurring in a prokaryotic oreukaryotic cell, in particular as regards the factors which influencethe binding of the functional nucleic acid to its target molecule.

In a preferred embodiment the body fluid is selected from the groupcomprising blood, urine, liquor (anatomical fluid), lymph fluid, serum,plasma, vaginal secretions, saliva and sperm.

In one embodiment the receptor is defined by its function in a cell.Accordingly the receptor can be selected from the group comprisingmolecular receptors, enzymes, metabolic intermediates, signal peptides,chaperone molecules and intracellular structures such as for exampleribosomes, mitochondria, elements of the cytoskeleton such as forexample tubulin and actin filaments, endosomal particles, lysosomes,other intracellular structures such as vesicles, in particularintracellular vesicles. As used herein the term molecular receptordenotes in a preferred embodiment a molecule which accepts informationand transmits this within a cell, a tissue, an organ or an organism. Theinformation is typically mediated by a molecule which interacts with themolecular receptor. As a result of the interaction the molecularreceptor is able to generate a signal. Such a signal can be based on thechange in the confirmation and/or of the activity of the receptor or canbe manifested therein. The signal itself is able to transmit in anotherform the information received or processed by it. As a result of thechange in the confirmation or activity of the molecular receptor, thesignal can preferably be a chemical, biochemical or an electricalsignal. Preferably the molecular receptor is part of a reaction cascade,and more preferably part of a signal cascade. The informationtransmitted by a molecular receptor can be quantitative and/orqualitative information, for example concerning the presence of acompound and/or its concentration.

In a preferred embodiment the term “metabolic intermediates” denotes allthose compounds which, due to metabolic activities in a cell, occur asconstituents of catabolism as well as of anabolism.

In a further embodiment the receptor is defined by its chemical nature.Preferably the receptor is selected from the group comprisingpolypeptides, carbohydrates, nucleic acids, lipids and combinationsthereof. As used herein, the term polypeptide preferably denotes anypolymer consisting of two or more amino acids. Preferably the aminoacids are L-amino acids, though D-amino acids may also be used withinthe scope of the embodiment. As used herein the term “nucleic acids”preferably denotes a polymer of two or more nucleotides or nucleotideanalogues which are known to the person skilled in the art in thisfield, wherein the nucleotides may be either D-nucleotides orL-nucleotides or mixtures thereof. Preferred combinations includeglycosylated polypeptides and glycosylated lipids.

A particular group of intracellular receptors are transcription factorsand DNA-binding proteins which bind to an AT hook. Examples oftranscription factors are given in the following table 1:

TABLE 1 Transcription factors gamma)OBP 14-3-3 epsilon 70-75K protein(STAT5A)4 14-3-3 zeta 80-90K protein 120-kDa CRE- 50-55K protein AAFbinding protein 53BP1 ABF-1 ADA2 AP-2alphaB Bach1t ADA3 AP-2beta Bach2ADA-NF1 AP-2gamma BAF155 AFP1 AP-2rep BAF47 AhR AP-3 (1) BAF53a AhR:Arnt AP-3 (2) BAF60A AIIN3 AP-4 Barhl1 Aiolos AP-5 Barhl2 AIRE APC Barx1AKNA AR Barx2 ALF Arnt Bcl-3 ALL-1 Arnt (774 AA form) BCL-6 alpha-CBFARNT2 beta-catenin alpha-CP2b ASC-2 Bin1 alphaH0 ASPP1 BMAL2alphaH2-alphaH3 ASPP2 B-Myb ALX3 ATBF1-A BNC Alx-4 ATBF1-B BP1 aMEF-2ATF BP2 AML1 ATF-1 BR140 AML1a ATF2 Brachyury AML1b ATF-2 BRCA1 AML1cATF-2: c-Jun BRCA2 AML1DeltaN ATF3 BRG1 AML2 ATF3 deltaZIP BRIP1 AML3ATF4 Brm AML3a ATF5 BTEB1 AML3b ATF6 BTEB2 AMY-1L ATF-a BTEB3 A-MybATF-adelta BTEB4 ANF ATOH1 B-TFIID AP-1 ATPF1 C/EBPalpha AP-2alphaABach1 C/EBPbeta C/EBPdelta CIITA CtBP2 C/EBPepsilon c-Jun CTCFC/EBPgamma c-Jun: JunD CTF CA150 CLIM1 CTF-1 c-abl CLIM2 CTF-2CACCC-binding factor CLOCK CTF-3 CAR c-Myb CTF-5 CAR: RXR-alpha c-MycCTF-7 Cart-1 C-Myc 1 CUP CBAF c-Myc: Max CUTL1 CBF (4) CNBP CUTL2 CBF(5) CoS Cx CBP COUP-TF1 cyclin A CCAAT- COUP-TF2 cyclin T1 bindingfactor CP1A cyclin T2 CCF CP1C cyclin T2a CCG1 CP2 cyclin T2b CCK-1aCPBP DAP CCK-1b CPE binding protein DAX1 CD28RC CREB DB1 Cdc5 CRE-BPaDBF4 cdk2 c-Rel DBP cdk9 c-Rel: RelA DbpA Cdx-1 CREMalpha DbpAv CDX2CREST DbpB Cdx-4 CRF DCoHm c-Ets-1 Crx DDB c-Ets-2 CSA DDB-1 CFF CSBDDB-2 c-Fos CSBP-1 DEC1 ChCh CSEN DEC2 CHOP-10 c-Ski DEF Chx10 CtBP1deltaCREB deltaFosB E2F EllaE-Cbeta deltaMax E2F + E4 EivF DeltaNp63beta E2F + p107 EKLF DeltaN p73alpha E2F-1 ELF-1 DeltaN p73betaE2F-1: DP-1 ELFR DeltaN p73gamma E2F-1: DP-2 elios DeltaNp63alpha E2F-2Elk-1 DeltaNp63gamma E2F-3a Emx-1 Dermo-1 E2F-4 Emx-2 DF-1 E2F-4: DP-1En-1 DF-2 E2F-4: DP-2 En-2 DF-3 E2F-5 ENH-binding protein Dlx-1 E2F-6ENKTF-1 Dlx-2 E2F-7 EP400 Dlx-3 E47 EPAS1 Dlx-4 (long isoform) E4BP4Epicardin Dlx-4 (short isoform) E4F epsilonF1 Dlx-5 E4F1 ER-alpha Dlx-6E4TF2 ER-alpha: ER-beta DP-1 E7; HPV-16, Papilloma ER-beta DP-2 Virustype 16 ER-beta1 DPBF EAR2 ER-beta2 DRIL1 EBF ER-beta3 DSIF EBP-80ER-beta4 DSIF-p14 EC2 ER-beta5 DSIF-p160 EF1 ERF DTF Egr-1 Erg-1 DUX1Egr-2 Erg-2 DUX2 Egr-3 ERM DUX3 Egr-4 ERR1 DUX4 EllaE-A ERR2 E EllaE-BERR3 E12 EllaE-Calpha ERR3-1 ERR3-2 FOXB1 FOXO1a ERR3-3 FOXC1 FOXO1bERRalpha1 FOXC2 FOXO2 ESE-1 FOXD1 FOXO3a ESE-1a FOXD2 FOXO3b ESE-1bFOXD3 FOXO4 ESE-2 FOXD4 FOXP1 ESE-2a FOXE1 FOXP2 ESE-2b FOXE3 FOXP3ESE-3 FOXF1 FOXP4 ESE-3a FOXF2 Fra-1 ESE-3b FOXG1a Fra-2 ESXR1 FOXG1bFTF ETF FOXG1c FTS Ets-1 deltaVII FOXH1 FXR Evi-1 FOXI1 FXR: RXR-alphaEVX1 FOXJ1a FXR-alpha EZF-2 FOXJ1b FXR-beta1 EZH1 FOXJ2 (long isoform)FXR-beta2 EZH2 FOXJ2 (short isoform) G factor F2F FOXJ3 G6 factor FAC1FOXK1 GAAP-1 factor 2 FOXK2a GABP FBP FOXK2b GABP-alpha f-EBP FOXK2cGABPB FEV FOXL1 GABP-beta1 Fgf3 FOXL2 GABP-beta2 FKBP59 FOXM1a GAFFKHL18 FOXM1b gammaCAAT FKHRL1P2 FOXM1c gammaCAC1 FKLF FOXN1 gammaCAC2Fli-1 FOXN2 GATA-1 FosB FOXN3 GATA-2 GATA-3 HAF HiNF-A GATA-4 HAND1HiNF-B GATA-5 HAND2 HiNF-C GATA-6 HB9 HiNF-D Gbx1 HDAC1 HiNF-D3 Gbx2HDAC2 HiNF-E GCF HDAC3 HiNF-P GCMa HDAC4 HIP1 GCN5 HDAC5 HIV-EP2 GCNF-1hDaxx Hlf GCNF-2 HDBP1 HLTF GF1 HDBP2 HLTF (Met123) GKLF Heat-inducingfactor HLX GLI1 HEB HMBP GLI2 HEB1-p67 HMG I GLI3 HEB1-p94 HMG I(Y)GLIS2 HEF-1B HMG Y GMEB-1 HEF-1T HMGB1 GR HEF-4C HMGB2 GR-alpha HEN1HMGI-C GR-beta HEN2 HMX1 GRF-1 HES-1 HNF-1alpha-A Gsc HES-2 HNF-1alpha-BGscl Hesx1 HNF-1alpha-C GT-IC Hex HNF-1beta-A GT-IIA Hey1 HNF-1beta-BGT-IIBalpha Hey2 HNF-1beta-C GT-IIBbeta HeyL HNF-3 H1TF1 HFH-1HNF-3alpha H1TF2 HIC-1 HNF-3beta H1TF2A Hic-5 HNF-3gamma H4TF-1 HIF-1HNF-4 H4TF-2 HIF-1 alpha HNF-4alpha HNF-4alpha1 HOXC11 IB1 HNF-4alpha2HOXC12 IBP-1 HNF-4alpha3 HOXC13 ICER-II HNF-4alpha4 HOXC4 ICER-IIgammaHNF-4alpha7 HOXC5 Id1 HNF-4gamma HOXC6 Id1H HNF-6alpha HOXC8 Id2 hnRNP KHOXC9 Id3 HOX11 HOXD10 Id3/Heir-1 HOXA1 HOXD11 IF1 HOXA10 HOXD12 IFI-16HOXA10 PL2 HOXD13 IgPE-1 HOXA11 HOXD3 IgPE-2 HOXA13 HOXD4 IgPE-3 HOXA2HOXD8 Ik-1 HOXA3 HOXD9 IkappaB HOXA4 Hp55 IkappaB-alpha HOXA5 Hp65IkappaB-beta HOXA6 HPX42B IkappaBR HOXA7 HrpF II-1 RF HOXA9A HSBP1IL-10E1 HOXA9B HSF IL-6 RE-BP HOXB1 HSF1 (long) II-6 RF HOXB13 HSF1(short) ING1 HOXB2 HSF2 ING1b HOXB3 HSF4a INSAF HOXB4 HSF4b IPCS-BFHOXB5 HSF4c IPF1 HOXB6 hsp56 IPF1: Pbx HOXB7 Hsp90 IRF-1 HOXB8 IA-1IRF-1: C/EBPbeta HOXB9 iASPP IRF-2 HOXC10 iASPP-RAI IRF-3 IRF-4 KR3 Lmo1IRF-5 KRF-1 Lmo2 IRF-6 KRN LMO3 IRF-7A KSR-1 LMX1A IRF-7B Ku autoantigenLMX1B IRF-7H Ku70 L-Myc-1(long form) IRF-8 Ku80 L-Myc-1 (short form)IRF-9 KUP L-Myc-2 irlB LAF-4 LUN-1 IRX-1 LANA; KSHV, Kaposi's LUN-2IRX2a sarcoma-associated LXR-alpha Irx-3 herpes virus LXR-alpha:RXR-alpha Irx-4 (herpes virus 8) LXR-beta ISGF-1 LBP-1 LXR-beta:RXR-alpha ISGF-3 LBP-1a Lyl-1 ISGF-3alpha LBP-1d M factor Isl-1alphaLBP-32 Mad1 ITF LBP-9 Maf ITF-1 LBX1 MafB ITF-2 LCR-F1 MafF JRF LEF-1MafG JunB LEF-1B MafG: MafG JunB: Fra-1 LF-A1 MafK JunB: Fra-2 LHX1MAML1 JunD LHX2 MASH-1 JunD: Fra-2 LHX3a Max kappaY FaKtor LHX3b Max1KBP-1 LHX5 Max2 KER1 LHX6.1a MAZ KER-1 LHX6.1b MAZi KLF15 LIT-1 MAZRKLF7 LITAF MBF1 Kox1 LKLF MBF2 MBF3 Miz-1 NERF-2 MBP-1 (1) MLX Net MBP-1(2) MM-1 NeuroD1 MBP-2 MondoA NEUROD-2 MDBP MOP3 NEUROD-3 MECP-2 MR NFIII-a MEF-2A MRF-2 NF III-c MEF-2B1 Msx-1 NF III-e MEF-2C Msx-2 NF-1MEF-2C/delta32 MTA1-L1 NF-4FA MEF-2C/delta8 MTB-Zf NF-4FBMEF-2C/delta8,32 MTF-1 NF-4FC MEF-2D00 mtTFA NF-AB MEF-2D0B Mxi1 NF-AT1MEF-2DA0 Myf-3 NF-AT1 MEF-2DA′0 Myf-4 NF-AT2 MEF-2DAB Myf-5 NF-AT2-alphaMEF-2DA′B Myf-6 NF-AT2-beta Meis-1 Myocardin, Splice Form NF-AT3 Meis-2a1 NF-AT4 Meis-2b MyoD NF-AT5 Meis-2c MyoD: E12 NfbetaA Meis-2d MyT1NF-CLE0a Meis-2e MZF-1 NF-CLE0b Meis-3 NC1 NFdeltaE3A Mel-18 NC2NFdeltaE3B Meox1 NCOR1 NFdeltaE3C Meox1a NCOR2 NFdeltaE4A Meox2 NCXNFdeltaE4B MHox (K-2) NELF NFdeltaE4C MIF-1 NERF Nfe MITF NERF-1a NF-EMIXL1 NERF-1b NF-E2 NF-E2 p45 NF-Y NRF NF-E3 NF-YA Nrf1 NFE-6 NF-ZcNRF-1 NF-Gma NF-Zz Nrf1: MafG NF-GMb NGN3 Nrf1: MafK NFI/CTF NHP-1 Nrf2NFIA NHP-2 Nrf2: MafG NFIB NHP3 Nrf2: MafK NF-IL-2° NHP4 NRF-2beta1NF-IL-2B Nkx2-1 NRF-2gamma1 NFIX Nkx2-2 Nrf3 NF-jun Nkx2-3 Nrf3: MafKNF-kappaB Nkx2-5 NRL NF-kappaB(-similar) Nkx2-8 NRSF NF-kappaB1 Nkx3-1NRSF Form 1 NF-kappaB1 precursor Nkx3-1 v1 NRSF Form 2 NF-kappaB2 Nkx3-1v2 NTF NF-kappaB2 (p49) Nkx3-1 v3 Nur77 NF-kappaB2 precursor Nkx3-1 v4NURR1 NF-kappaE1 Nkx3-2 OAZ NF-kappaE2 Nkx6-1 OC-2 NF-kappaE3 Nkx6-2OCA-B NF-MHCIIA Nmi Octa factor NF-MHCIIB N-Myc Octamer NF-muE1N-Oct-2alpha binding factor NF-muE2 N-Oct-2beta Oct-B1 NF-muE3 N-Oct-4oct-B2 NF-S NOR1 oct-B3 NF-X NOR1/MINOR OLIG2 NF-X1 NPA3 Oligo1 NF-X2NPAS1 Otx1 NF-X3 NPAS2 Otx2 NF-Xc NP-TCII Otx3 OZF Pax-3 Pbx1A: HoxD4p107 Pax-3A Pbx1a: IPF1 p130 Pax-3B Pbx1b p160MBP Pax-4a Pbx1B: HoxA5p28 Modulator Pax-5 Pbx1B: HoxB7 p300 Pax-6 Pbx1B: HoxB8 p38ergPax-6/Pd-5a Pbx1B: HoxC8 p40x; HTLV-I, T-cell Pax-7 Pbx1B: HoxD4Lymphotropic virus Pax-8 Pbx1b: PKNOX1 type I Pax-8a Pbx2 p45 Pax-8bPbx2: HoxB8 p49erg Pax-8c Pbx2: Hoxc6 p50: c-Rel Pax-8d Pbx2: PKNOX1 p53Pax-8e Pbx3a p55 Pax-8f Pbx3a: Hoxc6 p55erg Pax-9 Pbx3b p63 Pbx PC2p63alpha Pbx1 PC4 p63beta Pbx1: HoxB1 PC5 p63delta Pbx1: HoxB2 PCAFp63gamma Pbx1: HoxB3 PDEF p65delta Pbx1: HoxB4 PEA3 p73 Pbx1: HoxB5PEBP2alpha p73alpha Pbx1: HoxB6 PEBP2beta p73beta Pbx1: HoxB8 PGC-1p73delta Pbx1: PKNOX1 PITX1 p73epsilon Pbx1: Tcl3 PITX2 p73eta Pbx1aPITX2A p73gamma Pbx1A: HoxA5 PITX2A: Nkx2.5 p73kappa Pbx1a: Hoxb7 PITX2Bp73zeta Pbx1a: Hoxb8 PITX2B: Nkx2.5 Pax-1 Pbx1a: Hoxc6 PITX2C Pax-2Pbx1A: HoxC8 PITX2C: Nkx2.5 PITX3 POU5F1 PU.1 PKNOX1 POU5F1A PuF PKNOX2POU5F1B Pur factor PLAGL1 POU5F1C pX; HBV, Hepatitis B PLAGL2 POU6F1Virus PLZF PPAR-alpha PXR-1 PML PPAR-alpha: RXR- PXR-1: RXR-alpha PML-3alpha PXR-1: RXR-beta Pmx2a PPAR-beta PXR-2 Pmx2b PPAR-gamma1 R1 PNRPPAR-gamma2 R2 PO-B PPAR-gamma3 RAR-alpha Pontin52 PPAR-gamma4RAR-alpha: RXR-alpha POU1F1 PPUR RAR-alpha: RXR-beta POU2F1 PRRAR-alpha: RXR- POU2F2 PR A gamma POU2F2 (Oct-2.1) PR B RAR-alpha1POU2F2B pRb RAR-alpha2 POU2F2C PRDI-BF1 RAR-beta POU2F3 PRDI-BFcRAR-beta: RXR-alpha POU2F3, Isoform a Preb RAR-beta2 POU2F3, Isoform d1Prop-1 RAR-gamma POU2F3, Isoform d2 PROX1 RAR-gamma: RXR- POU3F1 PSE1alpha POU3F2 P-TEFb RAR-gamma1 POU3F2 (N-Oct-5a) PTF Rb: E2F-1: DP-1POU3F2 (N-Oct-5b) PTFalpha RBP60 POU3F3 PTFbeta RBP-Jkappa POU3F4PTFdelta Ref-1 POU4F1(l) PTFgamma RelA POU4F1(s) Pu box binding factorRelB POU4F2 Pu box binding factor REVERB-alpha POU4F3 (BJA-B)REVERB-beta RFX1 SHOX2b Smad7 RFX1: RFX2 SHOXa Smad8 RFX1: RFX3 SHOXbSMIF RFX2 SHP Sna RFX3 SIII-p110 SnoN RFX4 SIII-p15 Sox1 RFX5 SIII-p18Sox10 RFX5: RFXAP: RFXANK SIM1 Sox11 RFXANK SIM2 Sox12 RFXAP SIP1 Sox13RFX-B-delta5 Six-1 Sox14 RF-Y Six-2 Sox17 RORalpha1 Six-3 Sox18RORalpha2 Six-4 Sox2 RORalpha3 Six-5 Sox20 RORbeta Six-6 Sox21 RORgammaSKIP Sox3 Rox SLUG Sox4 RP58 Smad1 Sox5 RPF1 Smad2 Sox7 RPGalpha Smad2(437 Sox8 RREB-1 amino acids) Sox9 RSRFC4 Smad3 Sp1 RSRFC9 Smad3: Smad4Sp2 RVF Smad4 Sp3 RX Smad4delta3 Sp4 RXR-alpha Smad4delta4 Spi-BRXR-beta Smad4delta4-6 SPT16 RXR-gamma Smad4delta4-7 SRC-1 SAP-1aSmad4delta5-6 SRC-3 SAP-1b Smad4delta6 SRCAP SF-1 Smad5 SREBP-1a SHOX2aSmad6 SREBP-1b SREBP-1c TAF(II)100 TBX19 SREBP-2 TAF(II)125 TBX1ASRE-ZBP TAF(II)135 TBX1B SRF TAF(II)170 TBX2 SRF: SRF TAF(II)18 TBX20SRY TAF(II)20 Tbx22 SSRP1 TAF(II)250 TBX3 (722 Staf-50 TAF(II)250Deltaamino acids) STAT1 TAF(II)28 TBX3 (742 STAT1: STAT1 TAF(II)30 aminoacids) STAT1: STAT3 TAF(II)31 TBX4 STAT1 alpha TAF(II)55 TBX5 (longisoform) STAT1beta TAF(II)70-alpha TBX5 (short isoform) STAT2TAF(II)70-beta Tbx5: Nkx2.5 STAT3 TAF(II)70-gamma TBX6 STAT3: STAT3TAF-I TCF STAT4 TAF-II TCF-1 STAT5A TAF-L TCF17 STAT5B Tal-1 TCF-1ASTAT5B: STAT5B Tal-1beta TCF-1B STAT6 Tal-2 TCF-1C SXR TAR factor TCF-1DSXR: RXR-alpha tat; HIV-1, TCF-1E SYT Immunodeficiency virus TCF-1FTSR-alpha: type 1 TCF-1G T3R-alpha: RXR-alpha Tax; HTLV-I, T-cellTCF-2alpha T3R-alpha1 Lymphotropic TCF-3 T3R-alpha2 virus type I TCF-4T3R-beta1 T-bet TCF-4(K) T3R-beta2 TBP TCF-4B TAF(I)110 Tbr-1 TCF-4ETAF(I)48 TBR2 TEF TAF(I)63 TBX18 TEF-1 TEF-2 TFIIH-MO15 TRF (2) TEF-3TFIIH-p34 TRRAP TEF-5 TFIIH-p44 TWIST TEL1 TFIIH-p62 TxRE BP Tel-2aTFIIH-p80 TxREF Tel-2b TFIIH-p80: CAK UBF Tel-2c TFIIH-p90 UBP-1 Tel-2dTFII-I UEF-1 Tel-2e TFIIIA UEF-2 Tel-2f Tf-LF1 UEF-3 TFE3 Tf-LF2 UEF-4TFEB TFP-95 USF1 TFEB-A TGIF USF1: USF2 TFEC TGIF2 USF2 TFIIA TGT3 USF2bTFIIA-alpha/beta TIEG-1 Vav precursor (main form) TIF1a Vax-2TFIIA-alpha/beta TIF1g VDR precursor (subsidiary form) TIF2 VITF;Vaccinia virus/, TFIIA-gamma TLE1 Homo sapiens TFIIB TLX Vpr; HIV-1,TFIID TLX3 Immunodeficiency virus TFIIE TMF type 1 TFIIE-alpha TR2-11WBSCR14 TFIIE-beta TR2-5 WSTF TFIIF TR2-9 WT1 TFIIF-alpha TR4 WT1 ITFIIF-beta TRAP WT1 I -KTS TFIIH TREB-1 WT1 I-del2 TFIIH* TREB-2 WT1-KTS TFIIH-CAK TREB-3 WT1-del2 TFIIH-cyclin H TREF1 XBP-1 TFIIH-MAT1TREF2 XW, V YAF2 ZF1 ZNF174 YB-1 ZF2 ZNF-20 YEBP ZFP-37 ZNF-24 YL-1 ZFXZNF33a YY1 ZFY ZNF35 ZAC ZHX1 ZNF43 ZBP89 ZIC2 ZNF44 ZBP99 ZID ZNF45 ZEB(1124 AA) ZNF11a ZNF7 ZEB (1154 AA) ZNF124 ZNF76 ZER6 p52 ZNF133 ZNF83ZER6 p71 ZNF143 ZNF85

A further group of intracellular receptors are the intracellular targetmolecules listed in the following Table 2.

TABLE 2 Intracellular target molecules “long-chain” fatty Acetyl-CoAmalate-citrate Amyloid precursor protein acid CoA ligase synthaseAnkarin “major basic” protein Acetylglucosaminyl Arginase “mixedfunction” transferase Argininosuccinate oxygenase Acetylsperminesynthetase 11β-hydroxylase (EC deacetylase Argininosuccinate lyase1.14.15.4) Acetyl transcylase Aromatase 18-hydroxylase AconitaseArylsulfatase 1-acylglycerol-3- Actin Aspartate phosphate acyltransferase Adenosine deaminase aminotransferase 2,3- Adenosylhomocysteine Aspartate oxidosqualene lanosterol hydrolasetranscarbamoylase cyclase Adenosyl methionine ATPase 21-steroidhydroxylase decarboxylase ATP diphosphohydrolase (EC 1.14.99.10)Adenylate cyclase bcl-2 oncogene protein 24,28-sterol reductaseAdenylate deaminase Connective tissue- 3-hydroxybutyrate Adenylatekinase activating peptide dehydrogenase Adenylo-succinate lyaseC5a-inactivating 3-ketothiolase Adenylo-succinate factor3-β-hydroxysteroid synthase Calcitonin dehydrogenase Alanineaminotransferase Calmodulin (EC5.3.3.1) Aldolase Calpain I5′-nucleotidase Aldose reductase Calreticulin 8-oxoguanosine Alkalinephosphatase Carbamoyl phosphate deglycosylase Alcohol dehydrogenasesynthetase abl oncogene protein Amidophosphoribosyl Carbonate anhydraseAcetolactate synthase amine transferase Casein kinase 1 Acetylcholineesterase AMP Casein kinase 2 Acetyl-CoA carboxylase phosphodiestereraseCatalase Catechol Amyloid β/A4 protein Glycerol phosphatemethyltransferase Dihydrouracil acyltransferase Cathepsin dehydrogenaseGlycerol phosphate Cathepsin B and L Dioxygenase dehydrogenase cdc 10Dopamine monooxygenase Glycinamide cdc 13 p60 Dynenin ribonucleotide cdc2 p34 Elastase transformylase cdc 25 p80 Elastin GTP-binding proteinChaparonin Elongation factor Tu Haemoglobin A Cholesterol esteraseEndo-rhamosidase Haemoglobin A1 Cholesterol Enolase HaemoglobinBarcelona mono-oxygenase Enoyl-ACP-hydratase Haemoglobin Barts Citratesynthetase Enoyl-ACP-reductase Haemoglobin Beth Israel Clathrin etsoncogene protein Haemoglobin Bunbury Collagenase Ferritin HaemoglobinCochin-Port Cortisone dehydrogenase Ferrodoxin Royal crk oncogeneprotein Fatty acid synthetase Haemoglobin Cowtown Cyclin A and B fgroncogene protein Haemoglobin Cranston Cyclophilin fps oncogene proteinHaemoglobin Creteil Cytidine deaminase Fructose bisphosphate HaemoglobinD Cytidylate deaminase aldolase Haemoglobin D Cytochrome C peroxidaseFumarase Los Angeles Cytochrome P450 GABA aminotransferase Haemoglobin DPunjab Cytosine Galactosidase Haemoglobin F methyltransferase GelatinaseHaemoglobin Gower dbl oncogene protein Gelsolin Haemoglobin DefensinGlucophosphate isomerase Hammersmith Diacyl glycerol GlucosylceramideHaemoglobin Hiroshima acyltransferase galactosyl transferase HaemoglobinIndianapolis Dihydrofolate reductase Glutaminase Haemoglobin KansasDihydroorotatase Glutamine phosphoribosyl Haemoglobin KariyaDihydroorotate pyrophosphate Haemoglobin Kempsey dehydrogenaseamidotransferase Haemoglobin Kenya Haemoglobin LeporeHydroxymethylglutaryl- Myeloperoxidase Haemoglobin M CoA-reductaseMyofilament Haemoglobin M Hydroxymethylglutaryl- myristoyl transferaseHyde Park CoA-synthetase Na/K ATPase Haemoglobin M Iwate HydroxysteroidN-acetylglucuronidase Haemoglobin M Saskatoon dehydrogenaseNAD-dependent sterol-4- Haemoglobin Nancy Hypoxanthine-guanine-carboxylase Haemoglobin Philly phosphoribosyl transferase NADaseHaemoglobin Quong Sze IMP-dehydrogenase NADPH-dependent 3- HaemoglobinRaleigh Indole lyase oxosteroid reductase Haemoglobin Ranier Inositolphosphate Nexin Haemoglobin S phosphatase N-ras oncogene proteinHaemoglobin Sealy int-1 oncogene protein Nucleolus protein B23Haemoglobin Seattle Isocitrate lyase Nucleoside diphosphate HaemoglobinSt. Louis Kinin-forming enzyme kinase Haemoglobin St. Mande Ki-rasoncogene protein Ornithine Haemoglobin Titusville Lactate dehydrogenaseaminotransferase Haemoglobin Torino Lactoferrin Ornithine HaemoglobinWayne Laminin carbamoyltransferase Haemoglobin York Leukocyte elastaseOrnithine decarboxylase Haemoglobin Zurich Lipocortin Orotatedecarboxylase Ha-ras oncogene protein Lipoxygenase Orotate HexokinaseL-myc oncogene protein phosphoribosyl transferase Histaminase Lysozymep53 Histidine decarboxylase Malate dehydrogenase Peptidyl amidoglycolateHSP 27 Malate synthase lyase Hydropyrimidine Malonyl transacylasePeptidyl prolyl isomerase hydrolase Mannosidase PF4 Hydroxyacyl-CoA- metoncogene protein Phenylalanine dehydrogenase Methaemoglobin hydroxylaseHydroxymethylglutaryl Methionine Phosphatidate phosphatase CoA-splittingenzyme adenosyl transferase Phosphoenol pyruvate Phosphofructokinase mosoncogene protein carboxykinase Phosphoglucokinase rel oncogene proteintRNA synthetase Phosphoglucomutase Ribonucleotide reductase TropomyosinPhosphoglycerate kinase Ribose phosphate- Tryptophan synthasePhosphoglyceromutase pyrophosphate kinase Tubulin Phospholipase A2 Ricintropoelastin Tyrosine kinase Phospholipase C acid phosphataseUbioquinone reductase Phospholipase CG1 acid protease UPA PhospholipaseD Heavy meromyosin Uridine monophosphate Phospholipase Sserine/threonine kinase kinase Phosphoribomutase Spectrin Vitamin Kreductase Phosphoribosyl phosphate Spermine synthase wee-1 gene producttransferase Squalene epoxidase Xanthine dehydrogenase pim oncogeneprotein Squalene monooxygenase Xanthine oxidase Plasminogen activator-src oncogene protein Xylosyl transferase inhibitor Sterolmethyltransferase yes oncogene protein Porin suc 1 p13 α-actin pRB(retinoblastoma gene Succinyl-CoA -synthetase α-mannosidase product)Superoxide dismutase α-melogenin pRb retinablastoma gene Tartratedehydrogenase α-tubulin product Thioesterase β-actin ProperdinThioredoxin β-glucuronidase Prostaglandin synthase Thrombospondinβ-glycerophosphatase Protein kinase C Thromboxane-A2- β-ketoacyl-ACP-Purine nucleoside synthetase reductase phosphorylase Thymidylatesynthetase β-ketoacyl-ACP- Pyruvate dehydrogenase Transacylasesynthetase Pyruvate kinase Triose phosphate isomerase β-spectrin rafoncogene protein Triose phosphate β-tropomyosin dehyrogenase β-tubulin

A further particularly preferred group of intracellular receptors arethe HMG proteins, such as are described for example in the InternationalPatent Application PCT/EP96/00716, and in particular the HMGA proteins.As used herein the term HMGA proteins preferably denotes overall thefollowing proteins: HMGA1, HMGA1a, HMGA1b and HMGA2.

The HMGA proteins have a modular structure and each comprise threeDNA-binding domains, which are termed “AT hooks” and are shown as DBD1to DBD3 in FIG. 2, as well as a very acidic C-terminal region. It isobvious to the person skilled in the art that antagonists which bind toone of the “AT hooks” recognise not only the HMGA1 proteins and thus thetwo splice variants HMGA1A and HMGA1B (see FIG. 2), but also exhibitcross reactivity with similar DNA-binding molecules such as HMGA2. Apartfrom HMGA2, many further proteins also have sequences similar to the “AThooks” and form in each case further receptors. Such proteins are listedinter alia in Table 3:

TABLE 3 Column 1: Protein data bank - Access codes; Column 2: Proteindesignation Q9UKB0 Human HMG-Protein-R Q9UKY1 ZHX1_Human Zinc finger-and Homoeobox-Protein 1 P55198 AF17_HUMAN AF-17 Protein [MLLT6] Q59F28Human Trithorax Homologon (Fragment) Q6PJQ2 Human ZNF406 Protein(Fragment) Q75PJ9 Human ZFAT-1 Protein Q75PJ7 Human ZFAT-3 ProteinQ75PJ6 Human TR-ZFAT Protein Q9ULG1 Human KIAA1259 Protein Q9NUK2 HumanHypothetical Protein FLJ11314 Q9NTG6 Human Hypothetical ProteinDKFZp434B0616 Q8IX01 SFR14_HUMAN Presumed Splice Factor Q9H5J8 HumanHypothetical Protein FLJ23363 Q6I9Y6 Human MGC5306 Protein Q8IX01-2Splice Isoform 2 of Q8IX01 Q8IX01-3 Splice Isoform 3 of Q8IX01 Q8IX01-4Splice Isoform 4 of Q8IX01 Q15291 RBBP5_HUMAN Retinoblastoma-bindingProtein 5 (RBBP-5) P51608 MECP2_HUMAN Methyl-CpG-binding Protein 2Q6IPE2 Human FLJ12800 Protein Q6QHH9 Human Methyl-CpG-binding Protein 2,Isoform B Q9H8H4 Human Hypothetical Protein FLJ13629 Q7Z384 HumanHypothetical Protein DKFZp686A24160 O42043 ENK7_HUMAN HERV-K_1q23.3Provirus P61569 ENK16_HUMAN HERV-K_10p14 Provirus Q86VM3 Human MYBbinding Protein 1a [MYBBP1A] Q9UNW3 Human Coat Protein RIC-2 Q9BWE0Human REPIN1 Protein (Hypothetical Protein ZNF464) Q9ULL5 Human KIAA1205Protein Q9NZH2 Human Dhfr Oribeta-binding Protein RIP60 Q9NZI3 HumanLinens epithelium-containing growth factor p52 Q9NY27 Human RegulatorySub-Unit 2 of Proteinphosphatase-4 Q86U91 Human HMGA2/RAD51L1 Fusionprotein O95368 Human Transcriptional Coactivator p52 Q9P015 HumanHSPC145 (Mitochondrial Ribosome protein L15) Q5U071 Human HMG Protein‘box 2’ Q9H0Y1 Human Hypothetical Protein DKFZp564I206 Q6ZP45 HumanHypothetical Protein FLJ26517 P17096-2 Splice Isoform HMG-Y of P17096[HMGA1] Q9Y6X0 SETBP_HUMAN SET-binding Protein (SEB) [SETBP1] Q8TEK3DOT1L_HUMAN Histone-Lysine N-Methyltransferase Q8TEK3-2 Splice Isoform 1from Q8TEK3 [DOT1L] Q03164 HRX_HUMAN Zinc finger-Protein HRX (ALL-1)Q86YP1 Human Transcription factor MLL UPN96240 Q86YN9 HumanTranscription factor MLL UPN95022 Q03164-2 Splice Isoform 4P-18B fromQ03164 [MLL] P04920 B3A2_HUMAN Anion Exchanger Protein 2 Q59GF1 HumanAnion Exchanger-2 type a-variant Q8TAG3 Human SLC4A2 Protein Q6P391Human PSIP1 Protein O75475 Human Linens epithelium-containing growthfactor p75 Q9UEY6 Human Anion exchanger-2 type a [SLC4A2] Q9UEY5 HumanAnion exchanger-2 type b2 [SLC4A2] Q9UEY4 Human Anion exchanger-2 typeb1 [SLC4A2] Q9UER6 Human Transcriptional coactivator p75 O00256 HumanDFS70 P04920-2 Splice Isoform B1 of P04920 [SLC4A2] Q9BTB1 HumanHypothetical Protein MGC10561 Q9UKB0 Human HMG Protein-R O43167ZBT24_HUMAN Zinc finger- and BTB-domain-containing protein Q8N455 HumanZBTB24 Protein [ZBTB24] Q5TED5 Human Zinc finger-Protein 450 [ZNF450]Q96CK0 Human Zinc finger-Protein 653 Q96AS7 Human Zinc finger-Protein653 P51888 PRELP_HUMAN Prolargine Precursor Q5JPC9 Human HypotheticalProtein DKFZp667H216 Q6FHG6 Human PRELP-Potein Q6ZR44 Human HypotheticalProtein FLJ46672 Q8NEZ4 MLL3_HUMAN Myeloid/lymphoid-Leukaemia protein 3Homologon Q96AC6 KIFC2_HUMAN Kinesine-like Protein KIFC2 Q9C0H5K1688_HUMAN Protein KIAA1688 P52926 HMGIC_HUMAN HMG Protein I-C Q9UKV3ACINU_HUMAN Inductor of apoptotic Chromatin condensation Q59F82 HumanC21orf2-Protein variant Q5VYT7 Human OTTHUMP00000021181 Q96M56 HumanHypothetical Protein FLJ32810 Q69YJ6 Human Hypothetical ProteinDKFZp667N107 Q8NEY3 SPAT4_HUMAN Spermatogene-associated Protein 4 Q12809KCNH2_HUMAN Potassium Potential-controlled Ion channel Sub-family Q8IYY4Human protein similar to the DAZ-interacting protein 1 [DZIP1L] Q6ZN04Human Hypothetical Protein FLJ16544 Q5SXN7 Human Serologically definedcolon cancer antigen 3 Q8IVG2 Human KIAA2009 Protein (Fragment) [RKHD3]Q75VX8 Human KIAA2038 Protein (Fragment) [KIAA2038] Q12809-2 SpliceIsoform 2 von Q12809 [KCNH2]

Against this background the present invention also relates to L-nucleicacids and in particular spiegelmers, which are directed against any ofthe target molecules mentioned in Tables 1 to 3.

Since the L-nucleic acid is used as an intracellularly active agent, inparticular within a cell, in order to bind there to an intracellularreceptor, intracellularly different forms of the interactions betweenthe intracellular receptor and its interaction partners can beinfluenced. Depending on the type of interaction partners of theintracellular receptor, the intracellular use of L-nucleic acids thusenables interactions of proteins, nucleic acids, lipids, carbohydrates,or combinations of proteins, nucleic acids, lipids, carbohydrates withone another and between one another to be influenced.

In connection with the use according to the invention of a L-nucleicacid, in particular a spiegelmer, as intracellular agent and the methodfor binding an intracellular receptor, it should be noted that thispreferably relates to an in vitro application and to an in vitro method.

In connection with the use according to the invention of a L-nucleicacid, in particular a spiegelmer, for the production of a medicament forthe treatment and/or prevention of a disease and/or for the productionof a medicament for diagnostic purposes, the target molecule is anintracellular target molecule. In this connection the intracellulartarget molecule is one that is causily or non-causily involved in thedisease or illness to be prevented, treated or diagnosed, but in anycase its binding to a L-nucleic acid that binds specifically theretomeans that, in the case of a medicament, the disease is alleviated,prevented or cured, and/or in the case of a diagnostic agent the diseaseor a predisposition thereto can be established or diagnosed. As usedherein the concept of diagnosis include an initial diagnosis as well assubsequent diagnoses, in particular diagnoses or investigations in orderfor example to follow or to determine the progression of the disease orthe stages of the disease. It is within the scope of the invention thatthe target molecule is an intracellular receptor as described herein, inparticular a transcription factor, an intracellular target molecule oran HMG protein. Within the scope of the present invention it is mostparticularly preferred if the target molecule is presentintracellularly, i.e. within a cell, and the interaction having aninfluence on the disease and/or diagnosis takes place intracellularlybetween the L-nucleic acid and in particular the spiegelmer, and thetarget molecule, i.e. the receptor. It is also within the scope of thepresent invention if the target molecule is present outside a cell andthe interaction between the L-nucleic acid and in particular thespiegelmer, and the target molecule, i.e. the receptor, takes placeextracellularly.

The indications for use of the medicament produced using an L-nucleicacid, in which the nucleic acid is directed against an intracellulartarget molecule, follow for the person skilled in the art from theinvolvement of the intracellular target molecule in the respectivepathogenicity mechanism on which the indication is based. Thus, it isknown for example for HMGA proteins that these are associated withcarcinomas (inter alia of the breast, lungs, skin, thyroid) as well asleukaemias and lymphomas and other malignant tumours, such as inter aliasarcomas (rhabdomyosarcoma, osteosarcoma). Also, HMGA proteins areexpressed in many types of mesenchymal tumours, including inter aliahamartomas (breast and lungs), fatty tissue tumours (lipomas),pleomorphic adenomas of the salivary glands, uterine leiomyomas,angiomyxomas, fibroadenomas of the breast, polyps of the endometrium andatherosclerotic plaques. HMGA is an interesting therapeutic target.Blockade of HMGA could be a suitable starting point for controllingcancer and preventing its metastatic spread. As described in detailherein, L-nucleic acids directed against HMGA proteins are also suitablefor the diagnosis and/or treatment of virus diseases andarteriosclerosis on account of the involvement of HMGA proteins in theregulation of the transcription of a large number of viral genes or themarked expression of HMGA and in particular HMGA1 in the tissuesaffected by arteriosclerosis, which is associated with neointimal,vascular smooth muscle cells, macrophages and new blood vessels.

Although—as has been surprisingly found by the present inventors—nucleicacids, preferably L-nucleic acids and particularly spiegelmers, are ableas such to penetrate a phospholipid double membrane such as acytoplasmic membrane and then to be intracellularly functional in thesense of the specific interaction with the intracellular receptor, theeffectiveness of the infiltration of the L-nucleic acid can beinfluenced and in particular enhanced by the use of various techniques.These techniques include the use of chemical compounds or molecules aswell as the use of physical measures. Irrespective of the type, thesetechniques are herein generally referred to as delivery vehicles. It iswithin the scope of the present invention that the inventors havelikewise established that aptamers too exhibit this property, and likethe spiegelmers can similarly be used involved together with thecomposition according to the invention for basically the same purposes,applications and uses.

In the use of chemical compounds and molecules, a further distinction iswhether the nucleic acid needs to be modified or not for the delivery. Amodification for the purposes of using a delivery vehicle is generallynot necessary if the delivery vehicle is or comprises a vesicle, such asfor example in the case of liposomes, polypeptide vehicles,cyclodextrins, dendrimers, nanoparticles and microparticles, and alsopolyethyleneimine. A modification for the purposes of using a deliveryvehicle is on the other hand normally necessary if the delivery vehicleuses receptor-mediated endocytosis, fusogenic peptides, signal peptidesor lipophilic conjugates. The group of physical techniques includes inparticular electroporation and iontophoresis. It will be recognised thatfurther techniques for transporting a compound through a phospholipiddouble membrane such as a cytoplasmic membrane are known to the personskilled in the art in this field, which in principle are also suitablefor the transfer of a functional nucleic acid, such as for example anaptamer and/or a spiegelmer.

The individual delivery vehicles which can be used within the scope ofthe various aspects of the present invention will be described in moredetail hereinafter.

Liposomes consist of artificial cationic lipids such asN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium sulfate(DOTAP), in which the cationic groups interact with the negativelycharged nucleic acids and neutralise their anionic charge. The transporttakes place via endocytosis (PNAS, 93:11493-11498, 1996). However,cationic liposomes are cytotoxic, especially in higher concentrations,which restricts their use in vitro and in vivo (Biochem Biophys ResCommun, 197:818, 1993; Biochem Biophys Res Commun, 1372:55-68, 1998). Onthe other hand the amphiphilic pyridinium-based lipid SAINT-2 is anon-toxic formulation (Nucleic Acids Res, 29:2079-2087, 2001). Also,pH-sensitive liposomes are a possible alternative, which consist ofamphiphatic molecules such as cholesteryl hemisuccinate (CHEMS) anddioleyl phosphatidyl ethanolamine (DOPE) (J Pharmacol Exp Ther,297:1129-1136, 2001). Widely differing formulations of liposomes can befound in the review articles by Dass and Torchili (Drug Delivery,9:169-180, 2002; Nat Rev Drug Disc, 4:145-160, 2005).

With receptor-mediated endocytosis (RME) transport mechanisms which arealready present in the cell membrane are utilised. For this purpose thenucleic acid is coupled for example via a poly-L-lysine (PPL) linkercovalently to a transporter protein (“carrier” protein). The choice oftransporter protein depends in this connection on the ability to bind tospecific receptors of the cell membrane and to accumulate in the cell byendocytosis. A cell-specific transport can thus be realised. Forexample, an antisense phosphorothioate directed against c-myc could beintroduced into M-14 human melanoma cells (Anticancer Res, 17:29-35,1997). However, an effective transport by means of RME depends in thiscase not only on the affinity of the receptor for the ligand, but alsoon the limitation of the selected receptor as regards thecells—especially in vivo. Furthermore the selected ligand must beinactive or have an enhancing effect as regards the therapeutic result,in order to avoid a possible toxicity of the transport vehicle. Thus,the selection and the ubiquitous propagation of the selected receptor invivo is decisive for a successful RME-based transport. Moreover, asequestration of nucleic acids in endosomal compartments has beenobserved in RME-based transport, which would appear to make this methodnot very promising for an intracellular transport or an intracellularrelease or delivery. Most important of all, the coupling between thereceptor and nucleic acid must be chosen so that the function of one orother is not reduced (J Pharmaceutical Science, 92 (8):1559-1573, 2003).

Fusogenic peptides have been used to enable peptide-oligonucleotideconjugates to fuse with the cell membrane and thus effect the transportin the cell (Bioconjug Chem, 9: 466-475, 1998; Bioconjug Chem, 6:43-53,1995; Nucleic Acids Research, 25:2730-2736, 1997).

The selected import of nuclear proteins from the cytosol into thenucleus is mediated by short peptide sequences, which are termed nuclearlocalisation signals (NLS). Thus, various NLS peptide derivatives can beused in order to transport nucleic acids into the nucleus (BioconjugChem, 10:1005-1012, 1999; Bioconjug Chem, 10:598-606, 1999; BioconjugChem, 6:101-108, 1995). In addition there are also so-called signalimport peptides (IP), which can promote the cellular uptake of nucleicacids and could be derived for example from Kaposi's fibroblast growthfactor (K-FGF) (Adv Drug Deliv Rev, 44:35-49, 2000).

Vesicles similar to viral capsids can be formed by blocks ofpolypeptides, which can serve as possible transport vehicles for anintracellular transport (Nat Materials, 3(4):244-8, 2004).

The hydrophilic character of oligonucleotides and the anionicphosphodiester backbone reduce the cellular permeation. Lipophilicconjugates are therefore one possible way of increasing the ability ofoligonucleotides to bind to lipoproteins and thereby improveintracellular delivery. The conjugate that has been most thoroughlyinvestigated is cholesterol (Antisense and Nucleic Acid DrugDevelopment, 12:103-128, 2002).

Cyclodextrins are cyclic oligosaccharides, which have a centralhydrophobic cavity and multiple hydroxyl groups on the outer surface.Cyclodextrins have therefore already been used for the transport ofanti-sense oligonucleotides in human T cell lines (Antisense Res Dev,5:185-192, 1995) and have also been used in vivo for intracellulartransport and for intracellular release or delivery of immunogenic CpGsequences (Biochem Pharmacol, 52:1537-1544, 1996). A wide variety offormulations of cyclodextrins are given in the review article by Davisand Brewster (Nature Reviews Drug Discovery 3:1023-1035, 2004).

Dendrimers are highly branched macromolecules, which are composed ofrepetitive units of, typically, polyamides. The molecules carryfunctional groups such as primary amino groups on their surface, whichinteract with other molecules by electrostatic interaction. A complexstructure formation thus takes place rapidly and in a highlyreproducible manner, which leads to complexes of low cytoxicity (NucleicAcids Research, 28:4225-4231, 2000; Clin Cancer Res, 7:3606-3612, 2001).

Cyanacrylate nanoparticles have been tested since the beginning of the1990s for the release or delivery of oligonucleotides. The interactionof oligonucleotides with the nanoparticles takes place through ion pairsof the anionic charge of the oligonucleotides with various hydrophobiccations, principally with charged nanoparticles. Polyisohexylcyanoacrylate (PIHCA), polyisobutyl cyanoacrylate (PIBCA) or polyhexylcyanoacrylate (PHCA) are commonly used for the formation ofnanoparticles, although a large number of lipophiliccation-oligonucleotide pairs have also been tested (Pharm Res.,1:1370-1378, 1994; PNAS, 91:10460-10464, 1994; Pharm Res, 9:441-449,1992). Also, nanoparticles have already been employed for in vivo use(Biochem Biophys Res Commun, 279:401-406, 2000; Pharm Res, 13:38-43,1996).

Microparticles or so-called microspheres are typically formed frombiodegradable polymers such as poly(d,l-lactide-co-glycolides [P(LA-GA)]and are used for the delayed release of oligonucleotides (J Pharm Sci,91:790-799; 2000; J Controlled Release, 69:197-207, 2000; J Drug Traget,5:291-302, 1998).

Electroporation is a transport technology, which uses a strong electricfield in order to destabilise the lipid double membrane, and therebypermeabilise the cell membrane and thus effect a transport of thesubstance to be administered, which can also be present in ionised form,into the cell (iontophoresis). Electroporation has already beensuccessfully used in order to effect transdermal transport ofoligonucleotides ex vivo as well as in vivo (Int J Pharm, 184:147-156,1999; J Drug Traget, 5:275-289, 1998; Pharm Res, 15:1596-1602, 1998; IntJ Cancer, 85:260-266, 2000; Biochem Biophys Res Commun, 212:286-292,1995; Blood, 88:731-741, 1996).

The uptake of “naked” oligonucleotides into cells can be improved invitro and ex vivo by the use of high pressure.

The need for closed systems in order to use this technology means thatit can only be used for ex vivo applications (PNAS, 96:6411-6416, 1999;Hum Gene Ther, 10:2355-2664, 1999).

Also, the use of shockwaves, acoustic high pressure pulses, effects thetransport of oligonucleotides into cells (J Mol Med, 79:306-313, 2001;Cancer Res, 58:219-221, 1998). Ultrasound is an acoustic technologycomparable to shockwaves, but employs higher frequencies (MHz instead ofHz) and shorter application times (from seconds to minutes), and hasalready been used in a supporting role in gene therapy techniques (HumGene Ther, 7:1339-1346, 1996; Invest Radiol, 32:723-727, 1997;Ultrasound Med Bio, 25:1451-1457, 1999).

In a further aspect of the present invention a new delivery vehicle isprovided, which is suitable in particular for the transport offunctional nucleic acids such as aptamers, preferably functionalL-nucleic acids, and most particularly preferably spiegelmers. Thedelivery vehicle is in this case a micelle-like or liposome-likestructure based on polyethyleneimine. Without wishing to be too specificin the following description, the present inventors start from theassumption that the nucleic acid is present embedded or contained in themicelle-like or liposome-like structure. Polyethyleneimine can inprinciple be present and also used as linear or branchedpolyethyleneimine, polyethyleneimine in the branched form beingparticularly preferred. Moreover, polyethyleneimine can exist and canalso be used as high molecular weight or low molecular weightpolyethyleneimine. Preferably high molecular weight polyethyleneiminehas a molecular weight of about 800 kDa and low molecular weightpolyethyleneimine has a molecular weight of about 3 kDa. Within thescope of the present invention a polyethyleneimine with a mean molecularweight of about 25 kDa is preferred, a branched polyethyleneimine with amolecular weight of about 25 kDa being particularly preferred.

Although it is not essential for an effective implementation, it isnevertheless preferred if in the delivery vehicle according to theinvention the nucleic acid itself to be delivered also carries amodification. In this connection it is preferred if the modification isselected from the group comprising PEG residues. It is furthermorepreferred if the PEG residue has a molecular weight of about 1000 to10000 Da, preferably about 1200 to 5000 Da, more preferably about 1500to 2500 Da and most particularly preferably about 2000 Da.

When mixing the nucleic acid with the delivery vehicle to produce acomposition according to the invention, the ratio of the total number ofnitrogen groups of the polyethyleneimine to the total number ofphosphate groups of the nucleic acid to be delivered via or packagedwith the delivery vehicle is adjusted to about 1 to 20, preferably about1.5 to 10, more preferably 2 to 5, and most particularly preferablyabout 2 to 3.

The delivery vehicle according to the invention thus enables themechanism of intracellular transport of nucleic acids via condensationor packing with charged particles or reagents and associated change inthe charge of the overall complex, to be used also for functionalnucleic acids such as aptamers, and in particular L-nucleic acids suchas spiegelmers. This complex is readily taken up through endocytosis andthereby passes into the cytosol of the cell. A disadvantage of thismethod is the stability of the DNA/RNA and the release of the nucleicacid from the endosomal compartment. In the cytosol of the cell alysosome is rapidly formed from the tightly constricted endosome due tothe introduction of proteases or nucleases and by protonation of thecompartment. There nucleases break down the nucleic acids. This does notapply however to spiegelmers, since due to their unnatural configurationthese are nuclease-stable. Also, nucleic acids are not stable in theacidic environment of the lysosome. However, this is more true ofnucleic acids synthesised from DNA, and less true of nucleic acid fromRNA. The whole complex is rapidly transported out of the cell again byexocytosis and breakdown in the Golgi apparatus, and accordingly only afew nucleic acids pass into the cell. One of the challenges which asuitable transfection system has to overcome is thus the stabilisationas well as the release of the nucleic acid from the endosomes into thecytosol. As regards stability, RNA spiegelmers have ideal properties fora transfection of eukaryotic cells, since being enantiomers they are notsplit by enzymes.

The use according to the invention of L-nucleic acids and in particularin connection with the composition according to the invention isimportant specifically for this class of active substances, since theiraction mechanism is based on a stoichiometric approach and not on acatalytic approach, in which the intracellular release of just a fewmolecules is already sufficient to achieve the desired effect. To thisextent the present invention satisfies a need that was not hitherto metby the techniques of the prior art.

The transfection system according to the invention that is provided andelaborated by the delivery vehicles according to the invention is basedon the formation of micelles from nucleic acids and branchedpolyethyleneimine (PEI). The phosphodiester backbone of the nucleicacids interacts with the free nitrogen positions of the PEI and formssmall micelles through cross-linking, which have a positive charge onaccount of the PEI. These micelles are readily taken up as endosomesfrom a cell by constriction of the plasma membrane. The PEI now buffersinflowing protons, as a result of which many chloride ions in theinterior of the endosome lead to a swelling of the compartment onaccount of the osmotic pressure. This effect of PEI is described in theliterature as the proton sponge effect, and ultimately leads to therupture of the endosome and the release of the spiegelmers into thecytosol. (Pharm Res, 22 (3): 373-80, 2005; Eur J Cell Biol 83 (3):97-111, 2004; Gene Ther 9(24):1700-7, 2002).

It is within the scope of the present invention to apply the compositionaccording to the invention as an aerosol.

In addition spiegelmers can be derivatised with signal peptides forintracellular as well as intranuclear delivery, and also fororgan-specific delivery. A coupling of signal peptides directly to thepolyethyleneimine can be used for a targeted localisation in organs orwithin the cell.

In another further aspect the present invention relates to L-nucleicacids, in particular spiegelmers and more preferably RNA spiegelmers,which are directed against HMGA proteins. The spiegelmers disclosedherein directed against HMGA proteins are in particular examples of theknowledge, likewise forming the basis of the present invention, thatL-nucleic acids and in particular spiegelmers are able to overcome aphospholipid double membrane or a cytoplasmic membrane of a cell andbind intracellularly with the intracellular receptor, for the specificbinding to which they have been selected. As regards the configurationof the HMGA proteins and the L-nucleic acids directed against thelatter, the comments made herein regarding the intracellular use ofL-nucleic acids also apply in connection with the present aspect of theinvention (and vice-versa), and is referred to again at this point inorder to avoid unnecessary repetitions.

The HMG (high mobility group) family of DNA-binding phosphoproteins arepresent as non-histone components of chromatin throughout mammaliancells (Grosschedl et al. 1994). The basic HMG proteins are sub-dividedinto three different families—HMGB (formerly HMG-1/-2), HMGN (formerlyHMG-14/-17), and the HMGA family (formerly HMG-I/Y/C). Each HMG familyhas its characteristic functional sequence motif: the “HMG box” (HMGBfamily), the “nucleosomal binding domain” (HMGN family), and the “AThook” (HMGA family).

According to the current state of knowledge the HMGA family comprisestwo genes, HMGA1 and HMGA2. Three different proteins can be expressed byalternative splicing by HMGA1, (HMGA1a [formerly: HMG-I], HMGA1b[formerly: HMG-Y], HMGA1c [formerly: HMG-I/R]), whereas only one protein(HMGA2 [formerly: HMGI-C]), can be expressed by HMGA2. HMGA1a, HMGA1band HMGA2 are polypeptides of approximately 100 amino acid length andhave a modular sequence organisation: they possess three strongly basicregions (“AT hook”), which bind the narrow small channels ofdouble-stranded AT-rich DNA (Reeves & Nissen 1990). The C-terminus onthe other hand contains many acidic amino acids. The proteins do nothave a stable secondary structure when free in solution, and only adopta defined conformation when they are present in the complex with DNA orother proteins (Huth et al 1997). HMGA proteins belong to the moststrongly modified proteins in the mammalian cell nucleus and arephosphorylated, acetylated and methylated (Reeves & Beckerbauer 2001).

The HMGA proteins per se do not have any transcriptional activity, butbeing so-called architectonic transcription factors they organisethrough their protein-protein and protein-DNA interactions the formationof the nucleoprotein-DNA transcription complex (Wolffe 1994). They thusexert a regulatory activating or inhibitory influence on the expressionof a large number of genes. The most prominent example of a positiveregulation is the involvement of HMGA1 in the regulation of IFN-β(Thanos & Maniatis, 1992). Thus, for example in the case of the IFN-βpromoter HMGA1b stimulates the binding of NF-KB and ATF-2 to the DNAdouble helix and at the same time alters the DNA structure in such a waythat NF-KB and ATF-2 can interact with one another and presumably alsowith the rest of the transcription machinery (Thanos & Maniatis 1992, Duet al 1993). A further transcription-activating effect in connectionwith arteriosclerotic pathogenesis is the CD44 gene regulation inducedby HMGA1 (Foster et al 1998). CD44 is a cell surface glycoprotein and isinvolved in the migration and proliferation of smooth muscle cells afterendothelial damage (Jain et al 1996, Cuff et al 2001). Thetranscriptional regulation of CD44 is induced by the binding of c-Fosand c-Jun to the AP-1 binding site in the CD44 promotor and isstrengthened by the binding of HMGA1. Investigations in rats has shownthat due to CD44 over-expression, there is an intensified recruitment ofsmooth muscle cells, which has a direct influence on the formation ofarteriosclerotic lesions (Pellacani et al 1999; Foster et al. 1998;2000).

Investigations on the expression of the HMGA1 gene localised in thechromosomal band 6p21.3 and of the HMGA2 gene localised in the region12q14-15 showed that these are mainly active in processes of celldifferentiation.

Accordingly, a strong expression of these genes can be found duringembryo development and in undifferentiated cells (Chiappetta et al 1996)as well as in growth factor-stimulating cells (Friedman et al 1993;Johnson et al 1990; Ogram et al 1995; Holth et al 1997). In adult,differentiated tissue, HMGA1 is strongly expressed only in the retina,while HMGA2 is not found at all in the other tissues and HMGA1 is foundonly in very low concentrations (Bussemakers et al 1991; Chiappetta etal 1996; Rogalla et al 1996; Zhou et al 1995; Chau et al 2000). Areactivated expression of HMGA proteins in differentiated normal tissueis at the same time associated with the growth and differentiation ofadipocytes (Zhou et al 1995; Anand & Chada 2000; Melillo et al 2001),the proliferation of smooth muscle cells in the blood vessels aftervascular damage (Chin et al 1999), in the immune response ininflammatory reactions (Pellacani et al 1999), as well as in apoptoticprocesses (Diana et al 2001; Sgarra et al 2003). The amount of HMGA1varies in this connection depending on the proliferation rate of thecells (Johnson et al 1990).

During the course of embryo development the HMGA1 expression isconcentrated on specific organs of ectodermal, mesodermal or endodermalorigin, whereas HMGA2 is restricted to mesenchymal tissue. Up to now noinformation exists concerning the phenotype of HMGA1 knockout mice,possibly because the lack of this factor has damaged embryo developmenttoo severely. HMGA2 knockout mice on the other hand exhibit dwarfism andhave particularly little fatty tissue (Zhou et al 1995) and furthermoreare resistant to diet-induced obesity (Anand & Chada 2000).

Finally, HMGA2 and HMGA1b expression is not detectable in the fattytissue of normal mice, but is dramatically increased in the fat of fattyor diabetic mice (Chada et al. 2004), which points to a connectionbetween adiposity/obesity and HMGA expression.

Over-expression of HMGA1 influences in particular (Reeves et al 2001):

-   -   Cell cycle and growth regulators such as cdc25A,    -   Intermediary filament markers such as cytokeratin, type 1    -   Apoptosis regulators such as TRAR15    -   Oncogenes and tumour suppressor genes such as MET    -   Genes for DNA repair and recombination such as DNase X    -   Cell fate and development regulators such as frizzled-5    -   Receptors such as FGFR1    -   Cell adhesions, motility and invasion genes such as collagen        type 1    -   Angiogenesis regulators such as FGFR2    -   Invasion regulators such as MMP-16    -   Small GTPases of the Rho family and their regulators such as        RhoC    -   cell-cell interaction genes such as cadherin 12    -   Growth factors and cytokines such as IL-11

Abnormal regulation of HMGA1 could therefore lead to general alterationsof gene expression and thereby contribute significantly to the formationof transformed and/or metastatic phenotypes.

HMGA protein appear to play different roles in mesenchymal andepithelial tumours: in malignant epithelial tumours HMGA expression isassociated rather with later stages of carcinogenesis, whereas benigntumours—more often rarely converting mesenchymal tumours—already expressHMGA in early hyperplasia. This points to the fact that HMGA proteins intissues of different embryonic origin fulfil different functions, fromwhich also directly follows the corresponding uses of the L-nucleicacids according to the invention in the diagnosis and/or treatment ofcorresponding diseases, as is also illustrated in more detailhereinafter.

The expression of HMGA1 in various human and animal neoplasms wasinvestigated in animal models. The role of HMGA1 was demonstrated inanimal models of tumourigenesis (Leman et al 2003; Ram et al 1993) aswell as neoplastic progression (Bussemakers et al 1991; Nestl et al2001; Ram et al 1993).

Raised expression of the HMGA1 gene has been demonstrated in thefollowing carcinomas

-   -   Prostate (Bussemaker et al 1991; Tamimi et al 1996, Leman et al        2003; Nestl et al 2001)    -   Pancreas (Nestl et al 2001; Abe et al 2000, 2002; Tarbe et al        2001)    -   Thyroid (Chiappetta et al 1998, 1995)    -   Cervix (Bandiera et al 1998)    -   Stomach (Xiang et al 1997)    -   Breast (Holth et al 1997; Baldassarre et al 2003; Reeves et al        2001; Nestl et al 2001; Ram et al 1993; Dolde et al 2002)    -   Colon/Rectum (Fedele et al 1996; Abe et al 1999; Kim et al 1999;        Chiapetta et al 2001)    -   Ovaries (Masciullo et al 2003)        and furthermore in    -   Neuroblastoma (Giannini et al 2000; 1999) as well as    -   Lymphoma (Wood et al 2000a; b).

The precise reason for the increased expression and the role of theHMGA1 gene in the pathogenesis of the tumour and the process ofmetastasis has still not been fully clarified. Various studies indicatehowever that the strength of the HMGA1 expression by the respectivetumour as a prognostic marker correlates with its metastasing potentialand thus represents a characteristic feature of a malignant transformedcell (Giancotti et al 1987).

Further HMGA1-associated—in this case benign, mesenchymal tumours—arecharacterised by chromosomal changes in the chromosomal HMGA1 region6p21.3. Such aberrations have up to now been described inter alia in

-   -   Uterine leiomyoma (Mark et al 1988; Ozisik et al 1993)    -   Lipoma (Sreekantaiah et al 1990)    -   Endometrial polyps (Fletcher et al 1992; Dal Cin et al 1995) as        well as    -   chondroid hamartoma of the lungs (Fletcher et al 1991; Johansson        et al 1992, 1993).

Aberrations in the genetic mechanisms which control growth andproliferation are the primary cause of carcinogenesis. The expression ofHMGA proteins is strongly associated with tumour development, as hasbeen shown in a number of articles and papers (Giancotti et al. 1987,1989, 1993). Thus, a significant HMGA2 expression was found inchemically or virally caused tumours as well as in spontaneouslyoccurring tumours, whereas this protein could not be detected innon-transformed cells or healthy tissue (Giancotti et al. 1989). Inaccord with this, in the case of cells infected with oncogenicretroviruses in which the synthesis of HMGA2 expression had beenspecifically blocked, various phenotype markers for transformation wereabsent (Berlingieri et al. 1995).

The key role of HMGA proteins in normal as well as pathological growthhas been elucidated in mouse models: HMGA2 knockout mice exhibit stuntedgrowth, i.e. the animals are ca. 60% smaller than wild type mice. Thesedwarf mice however have a high resistance to chemically induced skintumours.

In the last few years structural aberrations of the chromosome region12q14-15 involving the HMGA2 gene have been found with the aid ofcytogenetic investigations for a whole number of benign tumours ofmesenchymal origin, these being the largest group of harmless neoplasiasin man. Despite a large number of aberrations (Schoenmakers et al 1995;Kottickal et al 1998; Klotzbüchel et al 1999) the altered formsnevertheless always exhibit a common feature: they retain all threeDNA-binding domains, but at the same time lose the acidic C-terminaldomain as well as, at the RNA level, the information of the 3′ UTR.

Such changes have already been found for many (mostly benign)mesenchymal HMGA-associated tumours:

-   -   Uterine leiomyomas, the most common abdominal tumours in women        and the reason for more than 200,000 hysterectomies per year in        the USA (Heim et al 1988; Turc-Carel et al 1986; Vanni et al        1988)    -   Lipomas (Heim et al 1988; Turc-Carel et al 1986; Mandahl et al        1987; Sreekantaiah et al 1991; Belge et al 1992)    -   Endometrial polyps (Walter et al 1989; Vanni et al 1993; Dal Cin        et al 1995)    -   Chondroid hamartomas of the lungs (Fletcher et al 1991, 1995;        Dal Cin et al 1993)    -   Pleomorphic adenomas of the salivary glands (Mark et al 1980,        1986; Bullerdiek et al 1987)    -   Haemangiopericytomas (Mandahl et al 1993)    -   Chondromatous tumours (Mandahl et al 1989; Bridge et al 1992)    -   Benign tumours of the breast (Birdsal at al 1992; Rohen et al        1995; Staats et al 1996)    -   Aggressive angiomyxomas (Kazmierczak et al 1995)    -   Diffuse astrocytomas    -   Osteoclastomas (Nuguera et al 1989)

The main cause of mortality and morbidity in cancer patients is themetastatic spread of the primary neoplasm in the body. Metastasis is nota simple process, since a successful colonisation of distant organs bydisseminated neoplastic cells has to pass through many stages.Neoplastic cells have to be released from the primary neoplasm, enterthe bloodstream, extravasate to distant sites, and finally proliferateagain in the parenchyma of the corresponding organ. Many genes whichexpress proteins such as proteases, adhesion molecules, motility factorsand angiogenic factors are involved in the various stages of this highlycomplex, metastatic cascade.

Which of these genes is ultimately decisive as regards metastisis is notknown. The HMGA1 gene, being one of the most important factorscontrolling this process, is however a likely candidate. The geneproducts of HMGA1 influence the transcription of many genes that areimportant for successful metastasis. For example, it has already beenshown that other metastasis-associated genes are themselves expressed ata reduced level in suppression of HMGA1 expression (Battista 1998;Vallone 1997).

HMGA1 is therefore an important therapeutic target molecule. Theblockade of HMGA1 is thus in principle suitable for controlling thecancer and preventing its metastatic spread (Evans 2004; Sgarra 2004).Thus for example, by using antisense RNAs directed against HMGAtranscripts, cell proliferation in cancer cells has been reduced invitro or the cells have even undergone apoptosis (Masciullo 2003; Scala2000; Chau 2003). It has been shown in animal models that the growth ofvarious pancreatic cancer xenografts is dramatically reduced by genetherapy (adoenoviral expression of antisense RNAs directed against HMGAtranscripts) (Trapasso et al 2004).

HMGA1 could furthermore be used as a prognostic diagnostic marker inorder to determine which patients would benefit from an aggressivecancer treatment. There is a close correlation between the degree of themalignant transformation and the amount of expressed HMGA1. This can inturn be correlated with a poor prognosis in many types of human cancer,such as prostate cancer (Tamimi 1996; Bussemakers 1991) and colorectalcarcinoma (Abe 1999) and neuroblastoma (Giannini 2000).

HMGA proteins are used by many viruses as well as by control factorsprovided by the host cell for the expression of viral genes or asco-factors, inter alia by

-   -   Human papovavirus JC (Leger et al 1995)    -   Epstein-Barr virus (Schaefer et al 1997)    -   Herpes simplex virus (Panagiotidis 1999; French et al 1996)    -   HIV-1 virus (Henderson et al 2000).

In particular HMGA proteins are involved in the regulation of thetranscription of a large number of viral genes in a host cell. Examplesof this are the regulation of the expression of the early and lateexpressed genes of the human papovavirus JC (Leger et al. 1995),regulation of the EBNA1 (Epstein-Barr virus nuclear antigen 1) gene ofthe Epstein-Bar virus (EBV), which is jointly responsible forcontrolling viral latency (Schaefer et al. 1997), regulation of the IE-3(immediate-early) gene of the Herpes simplex Virus-1 (HSV-1), whichcodes the prematurely expressed protein ICP4 (Panagiotidis et al. 1999),regulation of the promoter 2, active during the latency phase, of HSV-1(French et al. 1996) and regulation of the LTR (long terminal repeats)promoter of the humane HIV-1 virus (Henderson et al 2000).

The requisition of HMGA by the host cell in the context of viraldiseases is not only restricted to viral gene regulation. HMGA1 alsoappears to play a decisive role as architectonic co-factor in theintegration of the viral DNA of the HIV-1 virus, of the Moloney murineleukaemia virus (MoMuLv) and sarcoma bird flu virus (ASV) into the humangenome, and therefore appears to be an interesting therapeutic approachin antiviral treatment (Van Maele et al. 2006, Li et al 1998, Hindmarshet al. 1999).

Inhibitors of HMGA proteins are therefore also suitable for thetreatment and diagnosis of virus infections (Reeves & Beckerbauer 2002).

As a result of the previously demonstrated involvement of HMGA proteinsin various diseases and their suitability as diagnostic markers,L-nucleic acids and in particular spiegelmers directed against theseproteins can be used for the prevention, treatment and diagnosis of theabove diseases. Particularly preferred spiegelmers are in thisconnection the spiegelmers described herein. In this connection it isrecognised by those skilled in the art that although the individualspiegelmers have been developed for a specific HMGA protein, as a resultof the domain approach illustrated in Example 2 these also allow across-reactivity with other HMGA proteins, which can be seen from thealignment illustrated in FIG. 2.

Furthermore, it is recognised by those skilled in the art in this fieldthat the nucleic acids according to the invention contain a number ofstructural motifs, which define a class of spiegelmers that bind asintracellular receptors to HMGA proteins. The various structural motifsare illustrated in more detail in Example 1.

The nucleic acids according to the invention comprise in a preferredembodiment also those nucleic acids which are substantially homologousto the sequences specifically disclosed herein. The term “substantiallyhomologous” should preferably be understood in this connection to meanthat the homology is at least 75%, preferably 85%, more preferably 90%and most preferably more than 95, 96, 97, 98 or 99%.

The term nucleic acids according to the invention or nucleic acidsaccording to the present invention should furthermore be understood toinclude also those nucleic acids which comprises nucleic acid sequencessuch as are described herein, or parts thereof, preferably to the extentthat the nucleic acids or the said parts thereof are involved in thebinding to HMGA proteins. Such a nucleic acid can be derived from thosedisclosed herein, for example by shortening or truncation. A shorteningcan involve either one or both ends of the nucleic acids, as aredisclosed herein. A shortening can also involve the inner sequence ofnucleotides, i.e. can involve nucleotide(s) between the 5′ and the 3′terminal nucleotides. Furthermore the term shortening should also beunderstood as referring to the deletion of as few as one individualnucleotide from the sequence of the nucleic acids disclosed herein.Shortening can also involve more than one region of the nucleic acid(s)according to the invention, in which connection each of these regionsmay be as small as one nucleotide long.

The nucleic acids according to the present invention may furthermore beeither D-nucleic acids or L-nucleic acids. Preferably the nucleic acidsaccording to the invention are L-nucleic acids. In addition it ispossible that one or more parts of the nucleic acid is/are present asD-nucleic acids, or that at least one or more parts of the nucleic acidsis/are L-nucleic acids. The term “part” of the nucleic acids isunderstood to denote as little as one nucleotide. Such nucleic acids aregenerally referred to herein as D-nucleic acids or L-nucleic acids.

Accordingly, in a preferred embodiment the nucleic acids according tothe present invention consist of L-nucleotides and include at least oneD-nucleotide. Such a D-nucleotide is preferably fixed to a part that isdifferent from the region or regions that define the nucleic acidsaccording to the present invention, and is preferably fixed to thoseparts thereof which are involved in an interaction with other parts ofthe nucleic acids. Preferably such a D-nucleotide is fixed to the end ofeach region or to each nucleic acid according to the present invention.In a preferred embodiment such D-nucleotides can act as a spacer or alinker, which preferably binds modifications such as PEG and HES to thenucleic acids according to the present invention.

Within the scope of the present invention, in one embodiment the nucleicacids according to the invention also include those acids which are partof a longer nucleic acid, wherein these longer nucleic acids can includeseveral parts, at least one part being a nucleic acid according to thepresent invention or a part thereof. The other part or the other partsof these longer nucleic acids can either be a D-nucleic acid or aL-nucleic acid. Any combination can be used in conjunction with thepresent invention and for the purposes and uses such as have beendescribed herein for the nucleic acids according to the invention. Thisother part or these other parts of the longer nucleic acid can have afunction that is different from the binding function, and in particularfrom the binding to HMGA protein. A possible function is to allow aninteraction with other molecules, e.g. for the purposes ofimmobilisation, cross-linking, detection, amplification, modification orincreasing the molecular weight.

In particular in this connection L-nucleic acids as used herein arenucleic acids which consist of L-nucleotides, and preferably consistcompletely of L-nucleotides.

Accordingly, in particular D-nucleic acids as used herein are nucleicacids which consist of D-nucleotides, and preferably consist completelyof D-nucleotides.

Irrespective of whether the nucleic acid according to the inventionconsists of D-nucleotides, L-nucleotides or a combination of the two,the combination being for example a random combination or a definedsequence of regions which consist of at least one L-nucleotide and atleast one D-nucleic acid, the nucleic acid can consist of one or moredeoxyribonucleotides, ribonucleotides and combinations thereof.

In a further aspect the present invention relates to a pharmaceuticalcomposition which consists of at least one of the nucleic acidsaccording to the invention in combination with one or more other nucleicacids, in which the other nucleic acid(s) preferably binds to targetmolecules other than HMGA protein or exerts a function different to thatof the nucleic acids according to the invention.

The construction of the nucleic acids according to the invention asL-nucleic acids is advantageous for several reasons. L-nucleic acids areenantiomers of naturally occurring nucleic acids. D-nucleic acids arehowever not very stable in aqueous solutions and in particular inbiological systems and in biological samples, on account of theextensive presence of nucleases. Naturally occurring nucleases, inparticular nucleases from animal cells, are not able to break downL-nucleic acids. As a result of this the biological half-life of theL-nucleic acid in such a system, including the human and animal body, issignificantly increased. On account of the lack of degradability ofL-nucleic acids no nuclease breakdown products are produced and thus noresultant side effects are observed. This aspect in fact demarcatesL-nucleic acids from all other compounds that are used in the treatmentof diseases and/or disorders and include the presence of HMGA or itscausal involvement. L-nucleic acids that bind specifically to a targetmolecule through a mechanism different from the Watson-Crick basepairing, or aptamers which consist partly or completely of L-nucleicacids, in particular those parts of the aptamer that are involved in thebinding of the aptamer to the target molecule, are termed spiegelmers.

It is also within the scope of the present invention for the nucleicacids according to the invention to be in the form of single-strand ordouble-strand nucleic acids, regardless of whether they are present asD-nucleic acids, L-nucleic acids or D-L-nucleic acids, and whether theyare DNA or RNA. Typically the nucleic acids according to the inventionare single-strand nucleic acids, which on account of the primarysequence contain defined secondary structures and can therefore alsoform tertiary structures. The nucleic acids according to the inventionmay however also be double-stranded, in the sense that two strands whichare complementary or partly complementary to one another are hybridisedwith one another. This imparts stability to the nucleic acids, whichbecomes important particularly if the nucleic acid exists in thenaturally occurring D-form instead of the L-form.

The nucleic acids according to the invention can be modified. Suchmodifications can involve individual nucleotides of the nucleic acid andare well-known in the prior art. Examples of such a modification aredescribed inter alia in Venkatesan N. et al. (2003) Curr Med Chem.October; 10(19):1973-91; Kusser, W. (2000) J Biotechnol, 74: 27-38;Aurup, H. et al. (1994) Nucleic Acids Res, 22, 20-4; Cummins, L. L. etal, (1995) Nucleic Acids Res, 23, 2019-24; Eaton, B. E. et al. (1995)Chem Biol, 2, 633-8; Green, L. S. et al., (1995) Chem Biol, 2, 683-95;Kawasaki, A. M. et al., (1993) J Med Chem, 36, 831-41; Lesnik, E. A. etal., (1993) Biochemistry, 32, 7832-8; Miller, L. E. et al., (1993) JPhysiol, 469, 213-43. Such a modification may for example be an H atom,a F atom or a O—CH₃ group or NH₂ group at the 2′ position of anindividual nucleotide that is contained in the nucleic acid. Furthermorethe nucleic acid according to the present invention can include at leastone LNA nucleotide. In one embodiment the nucleic acid according to thepresent invention consists of LNA nucleotides, and preferably completelyof LNA nucleotides.

In one embodiment the nucleic acids according to the present inventioncan be a multi-part nucleic acid. A multi-part nucleic acid as usedherein is a nucleic acid that consists of at least two nucleic acidstrands. These at least two nucleic acid strands form a functional unit,the functional unit being a ligand for a target molecule. The at leasttwo nucleic acid strands can be derived from one of the nucleic acidsaccording to the invention either by cleavage of the nucleic acid inorder to produce two strands, or by synthesis from a nucleic acidcorresponding to a first part of the total nucleic acid, i.e. nucleicacid according to the invention, and a further nucleic acidcorresponding to the second part of the total nucleic acid. It isrecognised that cleavage as well as synthesis can be used in order toproduce a multi-part nucleic acid where more than the two strandsdescribed above by way of example can be present. In other words, the atleast two nucleic acid strands are preferably different from two strandsthat are complementary to one another and hybridise with one another,although a complementarity can exist to a certain extent between thevarious nucleic acid parts.

The present inventors have established that the nucleic acids accordingto the present invention have a very advantageous K_(D) value range ordissociation value range, and therefore a very advantageous bindingconstant. One way of determining the binding constant is to use anequilibrium binding assay, as is described in Example 1.

The K_(D) value of the nucleic acids according to the invention ispreferably less than 1 μM. A K_(D) value of about 1 μM should becharacteristic of a non-specific binding of a nucleic acid to a target.As will be recognised by those skilled in the art, the K_(D) value of agroup of compounds such as for example the nucleic acids according tothe present invention varies within a certain range. The K_(D) of about1 μM mentioned above is a preferred upper limiting value for the K_(D)value. The preferred lower limiting value for the K_(D) of nucleic acidsbinding the target molecule can be about picomolar or less. It is withinthe scope of the present invention for the K_(D) values of theindividual nucleic acids which bind to HMGA, preferably to lie withinthis range. Preferred ranges can be selected by choosing a first numberwithin this range and a second number within this range. Preferred uppervalues are 0.25 μM, 0.1 μM, and preferred lower values are 100 nM, 10nM, 1 nM and 0.05 nM.

The nucleic acids according to the invention preferably bind to HMGA1bat 37° C. in solution with a dissociation constant K_(D)<20 nM, asillustrated in Example 2.

The nucleic acids according to the present invention can be of arbitrarylength, provided that they are still able to bind to the targetmolecule. It is recognised in the prior art that specific lengths of thenucleic acids according to the present invention are preferred.Typically the length is between 15 and 120 nucleotides. It is alsorecognised by those skilled in the art that any whole number between 15and 120 is a preferred possible length for the nucleic acids accordingto the present invention. Preferred ranges for the length of the nucleicacids according to the present invention are lengths of about 20 to 100nucleotides, about 20 to 80 nucleotides, about 20 to 60 nucleotides,about 20 to 50 nucleotides and about 30 to 50 nucleotides.

In one embodiment the nucleic acids according to the invention arepresent in modified form. A particularly preferred form of modificationis PEGylation. In this, the modification of the nucleic acids accordingto the invention involves coupling with polyethylene glycol (PEG) orother groups.

On account of the high stability of the nucleic acids according to theinvention, in particular in the embodiment in which these exist asL-nucleic acids, it is possible to administer the nucleic acidsaccording to the invention directly to a patient requiring such atreatment. Preferably the nucleic acids according to the invention areprepared as a physiological solution for topical or systemicapplication.

Apart from the direct use of the nucleic acids according to theinvention for the treatment, prevention and diagnosis of the diseasesdescribed herein, these can be present or used individually or incombination with others in a pharmaceutical composition. Thepharmaceutical composition according to the present inventionaccordingly comprises at least one of the nucleic acids according to thepresent invention and preferably a pharmaceutically acceptable binder.Such a binder may be any known binder or one known in the field. Inparticular such a binder is any binder, as is described in connectionwith the production of the medicament, as disclosed herein. In a furtherembodiment the pharmaceutical composition includes a furtherpharmaceutically active agent. It is within the scope of the presentinvention for the medicament described herein to constitute thepharmaceutical composition as is described herein.

Preferably the pharmaceutical composition is intended for intravenousadministration. It is however also within the scope of the presentinvention for such pharmaceutical compositions to be administeredintramuscularly, intraperitoneally or subcutaneously. Otheradministration routes are orally or intranasally, in which connectionthat form of administration is preferred that is least invasive, but atthe same time retains the effectiveness of the pharmaceuticalcomposition and the pharmaceutically active agent.

The nucleic acids according to the invention are preferably contained assuch, or in connection with the pharmaceutical composition according tothe invention, dissolved in a pharmaceutically acceptable solvent. Suchsolvents are in particular those that are selected from the groupcomprising water, physiological saline, PBS or a glucose solution, inparticular a 5% glucose solution. Such a carrier can be for examplewater, buffer, PBS, glucose solution, preferably a 5% glucose solution(iso-osmotic), starch, sugars, gelatin or any other acceptable carriersubstance. Such carriers are generally known to those skilled in the artin this field.

It is within the scope of the present invention for the pharmaceuticalcomposition to contain at least one of the nucleic acids according tothe invention in its various embodiments, including, but not restrictedthereto, the nucleic acid as conjugate, as described herein.

In a further embodiment the medicament comprises a furtherpharmaceutically active agent. Such further pharmaceutical active agentsare for example protease inhibitors, proliferation inhibitors andangiogenesis inhibitors and/or agents that have a cytostatic effect.Alternatively or in addition, such a further pharmaceutically activeagent is a further nucleic acid according to the present invention.Alternatively, the medicament comprises at least one or more nucleicacids that bind to a target molecule that is different from HMGA, or hasa function that is different from one of the nucleic acids according tothe present invention.

The pharmaceutical composition according to the present invention can beused for the treatment, diagnosis and/or prevention of each of thediseases or disorders described herein.

In a further aspect the present invention relates to a method for thetreatment of a living organism requiring such a treatment, wherein themethod includes the administration of a pharmaceutically active amountof at least one of the nucleic acids according to the present invention.In one embodiment the living organism suffers from a disease, or thereis a risk that it will suffer from such a disease, the disease being oneof those mentioned herein, in particular a disease that is described inconnection with the use of one of the nucleic acids according to thepresent invention for the production of a medicament.

Although the use of the nucleic acids according to the invention alreadyfollows from the involvement illustrated above of HMGA proteins in thevarious diseases and states, this aspect will be discussed furtherhereinafter for illustrative purposes.

HMGA proteins and their genes have in particular become increasinglyinvolved in the diagnosis and prognosis of neoplastic diseases and havebeen proposed as potential biomarkers. In healthy tissue the expressionlevel of HMGA1a/b proteins is very low, if detectable at all. RaisedHMGA1a/b protein expression is characteristic of the phenotype of alarge number of tumours and metastases of very many types of cancer(Sarhadi et al. 2006, Balcercak et al. 2005, Briese et al. 2006, Changet al. 2005, Peters et al. 2005, Sato et al. 2005, Chiappetta et al.2004, Li et al. 2004, Chuma et al. 2004, Donato et al. 2004, Czyz et al.2004, Kettunen et al. 2004, Lee et al. 2004, Chen et al. 2004, Abe etal. 2003, Blacerczak et al. 2003, Flohr et al. 2003, Masciullo et al.2003, Nam et al. 2003, Pierantoni et al. 2003). High HMGA proteinexpression correlates significantly with a poor prognosis and theformation of metastases. The detection of the HMGA1a/b expression levelin biopsies and its histological characterisation is a diagnosticapproach to the early detection, prognosis and identification ofneoplastic diseases, in particular the diseases and conditions discussedhereinbefore.

Furthermore an association between HMGA1 proteins and arterioscleroticplaques is described in the literature (Schlueter et al. 2005.). HMGA1regulates CD44, one of the principal target genes for the formation ofplaques. In this connection it was found, compared to the surroundingtissue, that the affected regions such as neo-intimal, vascular smoothmuscle cells, macrophages and new blood vessels have a high expressionof HMGA1. HMGA1 appears therefore to be one of the mediators in theformation of plaque and is thus a target molecule for diagnosticpurposes.

The L-nucleic acids described here and in particular the spiegelmers,which bind HMGA1a/b, can within the scope of the methods known to theperson skilled in the art be used in a similar way to antibodies. Up tonow only very few specific (differentiating) and affine antibodiesagainst HMGA1 have been identified and are commercially obtainable. Thisappears to be due to the non-existent secondary structure of HMGA1,which is not a suitable target for the MHC complex in the generation ofantibodies.

Against this background it was however surprisingly found that thebiotinylated HMGA1a/b-binding spiegelmer 5′-bio-NOX-A50 recognises inthe western blot procedure HMGA1a/b as individual bands in cancer celllines. Furthermore, as described in Example 2, recombinantly expressedHMGA1b protein could be detected. The detection of the biotinylatedspiegelmer is carried out for example by an anti-biotin antibodyconjugated by means of horseradish peroxidase (HRP).

The in vivo diagnosis of HMGA1a/b is a further approach, in which thenucleic acids according to the invention can be used. Tumours andmetastases are often embedded in necrotic tumour cells, which releaseHMGA1a/b to the surrounding tissue. The detection of the extracellularHMGA1a/b is one approach to the diagnosis of tumours and metastasesembedded in healthy tissue.

As preferably used herein, a diagnostic tool or diagnostic agent ordiagnostic means is able to detect either directly or indirectly an HMGAprotein, preferably HMGA1a/b, as described herein, and preferablyHMGA1a/b as described herein, in connection with the various disordersand diseases. The diagnostic tool is suitable for detecting and/orsearching for any of the diseases and conditions described herein. Sucha detection is possible by the binding of the nucleic acids according tothe present invention to HMGA1a/b. Such a binding can be detected eitherdirectly or indirectly. The corresponding methods and means are known tothose skilled in the art in this field. The nucleic acids according tothe present invention can inter alia be labelled, which permits thedetection of the nucleic acids according to the present invention,preferably the nucleic acid that is bound or can bind to HMGA proteinand preferably HGMA1a/b. Such a labelling is preferably selected fromthe group comprising radioactive, enzymatic and fluorescence labelling.In principle all known tests that have been developed for antibodies canbe adapted to the nucleic acids according to the present invention, thetarget molecule-binding antibody being replaced by a targetmolecule-binding nucleic acid. In antibody tests which employ unlabelledtarget molecule-binding antibodies, the detection is preferablyperformed with a secondary antibody, which has been modified withradioactive, enzymatic or fluorescence labels and binds to the targetmolecule-binding antibody at its Fc fragment. In the case of a nucleicacid, preferably a nucleic acid according to the present invention, thenucleic acid is modified with such a label, the said label preferablybeing selected from the group consisting of biotin, CY-3 and CY-5, andsuch a label is detected by an antibody directed against such a label,for example an anti-biotin antibody, an anti-CY-3 antibody or ananti-CY-5 antibody, or in the case where the label is biotin, the labelis detected by streptavidin or avidin, which naturally binds to biotin.Such an antibody, i.e. streptavidin or avidin, is in turn preferablymodified with a corresponding label, for example a radioactive,enzymatic or fluorescence label, similarly to a secondary antibody.

In a further embodiment the nucleic acids according to the presentinvention are detected or analysed by a second detection agent, thisdetection agent being a molecular beacon. The technique of molecularbeacons is known to those skilled in the art in this field. In brief,these molecular beacons are nucleic acid probes which are a reversecomplement of the nucleic acid probe to be detected, and accordinglyhybridise with a part of the nucleic acid probe to be detected. Afterthe binding of the nucleic acid probe the fluorophore groups of themolecular beacon are separated from one another, which leads to a changein the fluorescence signal, preferably a change in intensity. Thischange correlates with the amount of nucleic acid probe that is present.

It is within the scope of the present invention that the nucleic acidsaccording to the invention can appropriately be used as L-nucleic acidswithin the scope of the various aspects disclosed herein.

The nucleic acids according to the invention can furthermore be used asstarting material for the design of pharmaceutical active substances(drug design). In principle there are two possible approaches to thisproblem. One approach consists in screening libraries of compounds,wherein such libraries of compounds are preferably libraries of lowmolecular weight compounds (low or small molecules). Such libraries areknown to those skilled in the art in this field. In one embodiment thescreening is a high throughput screening. Preferably high throughputscreening is fast, efficient, and is carried out as a trial-and-errorevaluation of active substances in a target molecule-based assay.

Alternatively, according to the present invention the nucleic acids canbe used for the rational design of active substances. Preferably therational design of active substances is the design of a pharmaceuticalactive substance candidate. Starting from the three-dimensionalstructure of the target molecule, which is normally determined bymethods such as X-ray structure analysis or nuclear magnetic resonancespectroscopy (NMR), computer programs are used to search through databanks containing structures of a large number of different chemicalcompounds. The selection is carried out by computer. The selectedcompounds are in addition tested in the laboratory.

The rational design of active substances can take as its starting pointany of the nucleic acids according to the present invention, andcomprises a structure, in particular a three-dimensional structure,which is similar to the structure of the nucleic acid(s) according tothe invention or is identical to that part of the structure of thenucleic acid(s) according to the invention that mediates the binding toHMG proteins. In any case, such a structure also exhibits the same or atleast a similar binding behaviour to the nucleic acid(s) according tothe invention. In either a further step or as an alternative step, inthe rational design of active substances the preferablythree-dimensional structure of those parts of the nucleic acids bindingto HMG proteins is imitated by chemical groups, which are preferablydifferent to nucleotides and nucleic acids. By means of this imitation,also termed mimicry, a compound can be constructed which is differentfrom the nucleic acid or the nucleic acids which was/were used asstarting materials for the rational design of the active substance. Sucha compound or active substance is preferably a small molecule or apeptide.

In the case of screening libraries of compounds using competitive testswhich are known to those skilled in the art in the field, suitable HMGanalogues, HMG agonists and HMG antagonists can be found. Suchcompetitive assays can be designed as follows. The nucleic acidaccording to the invention, preferably a spiegelmer, i.e. a L-nucleicacid binding the target molecule, is coupled to a preferably solidphase. In order to identify HMG analogues, a labelled HMG protein isadded to the test system. Alternatively, the HMG protein could also becoupled to a solid phase and the nucleic acid according to the inventioncould be labelled. A potential analogue or a potential agonist orantagonist would compete with the HMG molecules which bind to thespiegelmer, which would result in a decrease in the signal received fromthe corresponding label. The screening for agonists or antagonists caninclude the use of a cell culture test system which is known to thoseskilled in the art in the field.

In a further aspect the nucleic acids according to the invention can, onaccount of their characteristic binding behaviour to HMG protein, beused for target (target molecule) validation. The nucleic acidsaccording to the invention can be used in an ex vivo organ model inorder to study the function of HMG protein. In principle there exist exvivo models in which HMG agonists/antagonists can be tested.

A kit according to the present invention can comprise at least one ormore of the nucleic acids according to the invention. In addition thekit can include at least one or more positive or negative controls. HMGprotein against which the nucleic acid according to the invention hasbeen screened, or to which this binds, preferably in liquid form, can beused as positive control. As negative control there can be used interalia a peptide that behaves as regards its biophysical propertiessimilarly to HMG protein, but which is not recognised by the nucleicacids according to the invention, or a peptide can be used having thesame amino acid composition but a different sequence to HMG protein.

Furthermore the kit can include one or more buffers. The variousconstituents can be present in the kit in dry or lyophilised form, ordissolved in a liquid. The kit can include one or more containers, whichin turn can contain one or more of the constituents of the kit.Preferably the vessels contain reaction batches, such as are necessaryfor a single execution of an experiment using one or more constituentsof the kit.

It will be acknowledged that, unless stated to the contrary, thesequences listed herein are given in the 5′-3′ direction. It willfurthermore be seen that the term “the two sections hybridise with oneanother” is understood herein to mean that the sections can hybridise invitro on the basis of general base pairing rules, or that the sectionshybridise or can hybridise under the conditions of use, but are notnecessarily hybridised with one another or are present in hybridisedform under the conditions of use.

The various SEQ. ID., the chemical structure of the nucleic acids asdisclosed herein and the target molecule HMGA1a/1b as used herein, theactual sequences and the internal references are summarised in thefollowing table.

Internal Seq. ID Reference RNA/Peptide Sequence 1 132-C3, NOX-h L-RNAGCUGCUGCAAAUUGACGGGGGCGUGGUUGGGGCGGGUCGAUUGCAGC (spiegelmer) 2132-B3, NOX-f L-RNA GCUGAAUGAGGAUCGCAGGGGCGUGGCUGGGGUGGGCGACCGUUCAGC(48nt) (spiegelmer) 3 132-C4 L-RNAGCUGCGCAAGGAGAGGGGCGCGGUUGGGGAGGCUCUAAGCGCUGCAGC (spiegelmer) 4 132-E2L-RNA GCUGGCGCUAUAGGACAGGGGUGCGGUUGGGGCGGUCCGCUGUCAGC (spiegelmer) 5132-A2 L-RNA GCUGGAUAGAACGCAGGGGUGCGGUUUGGGGUGGGCGUGAUAUGCAGC(spiegelmer) 6 132-H1, NOX-I L-RNAGCUGCCGUAAAGAGGGGUGAGGUUGGGGAGGCUUUACGGUUUCAGC (spiegelmer) 7 132-F1L-RNA GCUGCAUGCCGCGAUCAGGGGAGCGGUUGGGGCGGGAUCCGGCUCAGC (spiegelmer) 8132-G2, NOX-g L-RNA GCUGCGAGGGAGGUAGCGGCUCUGCGCCGUGACGUGGGUGGAUGCAGC(spiegelmer) 9 122-A1, NOX-A L-RNAGGCUGAUACGUGGGUGGAUAUGGGGCAGUUCCAUGUGGGUGGUUUCAGCC (spiegelmer) 10122-C1, NOX-B L-RNA GGCUGAUACGUGGGUGAAUAUGGGGCAGUUCCAUGUGGGUGGUUUCAGCC(spiegelmer) 11 122-B2 L-RNAGGCUGAUACGUGGGAGGAAAGGUGUAACUACCUGUGGGAGGUUUCAGCC (spiegelmer) 12122-E2, NOX-C L-RNA GGCUGGCACUCGCAGGGGUGAAGUGAUGAUUGGGGUGGGCGAGACCAGCC(spiegelmer) 13 122-G2, NOX-E L-RNAGGCUGCCGAGUGGUUGGGUGGUGUAAGGGAGGUGGAAUCCGCGGGCAGCC (spiegelmer) 14122-B4, NOX-D L-RNA GGCUGUUCGUGGGAGGAAGGCUCUUGGAUAGAGUCGUGGGUGGUUCAGCC(spiegelmer) 15 132-B3 32nt, L-RNA GGAUCGCAGGGGCGUGGCUGGGGUGGGCGACCNOX-f 32nt (spiegelmer) 16 132-B3 33nt, L-RNAGGAUCGCAGGGGCGUGGCUGGGGUGGGCGAUCC NOX-f 33nt (spiegelmer) 17HMGA1a/b target D-peptide Biotin-EPSEVPTPKRPRGRPKGSKNK molecule domain,Biotinyl-D- HMGA1a/b-21 mer 18 HMGA1a (human) L-peptide(M)SESSSKSSQPLASKQEKDGTEKRGRGRPRKQPPVSPGTALVGSQKEPSEVPTPKRPRGRPKGSKNKGAAKTRKTTTTPGRKPRGRPKKLEKEEEEGISQESSEEEQ 19 HMGA1b (human)L-peptide(M)SESSSKSSQPLASKQEKDGTEKRGRGRPRKQPPKEPSEVPTPKRPRGRPKGSKNKGAAKTRKTTTTPGRKPRGRPKK-LEKEEEEGISQESSEEEQ 20 HMGA2 human L-peptide(M)SARGEGAGQPSTSAQGQPAAPAPQKRGRGRPRKQQQEPTGEPSPKRPRGRPKGSKNKSPSKAAQKKAEATGEKRPRGRPRKWPQQVVQKKPAQEETEETSSQESAEED 21 bio-dsDNA D-DNA5′biotin-TCGAAAAAAGCAAAAAAAAAAAAAAAAAACTGGC)and 50 (AT hook)5′GCCAGTTTTTTTTTTTTTTTTTTGCTTTTTT 22 NOX-A-3′PEG, L-RNAGGCUGAUACGUGGGUGGAUAUGGGGCAGUUCCAUGUGGGUGGUUUCAGCC-2 kDA-PEGNOX-A-3′PEG2000, NOX-A-2 kDa PEG, NOX-A PEG 23 INVERSE-3′-PEG L-RNACCGACUUUGGUGGGUGUACCUUGACGGGGUAUAGGUGGGUGCAUAGUCGG-2 kDA-PEG INV 3′-PEG24 5′-biotin-NOX-A L-RNABiotin-GGCUGAUACGUGGGUGGAUAUGGGGCAGUUCCAUGUGGGUGGUUUCAGCC 255′-biotin-NOX-A L-RNABiotin-CCGACUUUGGUGGGUGUACCUUGACGGGGUAUAGGUGGGUGCAUAGUCGG inverse 26INVERSE L-RNA CCGACUUUGGUGGGUGUACCUUGACGGGGUAUAGGUGGGUGCAUAGUCGG 27POC-3′-PEG L-RNA UAAGGAAACUCGGUCUGAUGCGGUAGCGCUGUGCAGAGCU-2 kDA-PEG 28Capture probe L-RNA CCCATATCCACCCACGTATCAGCCTTTTTTTT-NH₂ NOX-A 29Detector probe L-RNA Biotin-TTTTTTTTGGCTGAAACCACCCACATGG NOX-A 30Capture probe L-RNA NH₂(C7)-TTTTTTTTTAGCTCTGCACAGCGCT POC 31Detector probe L-RNA CCGCATCAGACCGAGTTTCCTTATTTTTTTT-Biotin POC 32HMG_fwd1 Primer D-DNA TCGACACCATGGGTGAGTC 33 HMG_rev1 Primer D-DNAGTCTAGAAAGCTTCCCAACTG 34 132-C3, NOX-h D-RNAGCUGCUGCAAAUUGACGGGGGCGUGGUUGGGGCGGGUCGAUUGCAGC (aptamer) 35132-B3, NOX-f D-RNA GCUGAAUGAGGAUCGCAGGGGCGUGGCUGGGGUGGGCGACCGUUCAGC(48nt) (aptamer) 36 132-C4 D-RNAGCUGCGCAAGGAGAGGGGCGCGGUUGGGGAGGCUCUAAGCGCUGCAGC (aptamer) 37 132-E2D-RNA GCUGGCGCUAUAGGACAGGGGUGCGGUUGGGGCGGUCCGCUGUCAGC (aptamer) 38132-A2 D-RNA GCUGGAUAGAACGCAGGGGUGCGGUUUGGGGUGGGCGUGAUAUGCAGC (aptamer)39 132-H1, NOX-i D-RNA GCUGCCGUAAAGAGGGGUGAGGUUGGGGAGGCUUUACGGUUUCAGC(aptamer) 40 132-F1 D-RNAGCUGCAUGCCGCGAUCAGGGGAGCGGUUGGGGCGGGAUCCGGCUCAGC (aptamer) 41132-G2, NOX-g D-RNA GCUGCGAGGGAGGUAGCGGCUCUGCGCCGUGACGUGGGUGGAUGCAGC(aptamer) 42 122-A1 , NOX-A D-RNAGGCUGAUACGUGGGUGGAUAUGGGGCAGUUCCAUGUGGGUGGUUUCAGCC (aptamer) 43122-C1, NOX-B D-RNA GGCUGAUACGUGGGUGAAUAUGGGGCAGUUCCAUGUGGGUGGUUUCAGCC(aptamer) 44 122-B2 D-RNAGGCUGAUACGUGGGAGGAAAGGUGUAACUACCUGUGGGAGGUUUCAGCC (aptamer) 45122-E2, NOX-C D-RNA GGCUGGCACUCGCAGGGGUGAAGUGAUGAUUGGGGUGGGCGAGACCAGCC(aptamer) 46 122-G2, NOX-E D-RNAGGCUGCCGAGUGGUUGGGUGGUGUAAGGGAGGUGGAAUCCGCGGGCAGCC (aptamer) 47122-B4, NOX-D D-RNA GGCUGUUCGUGGGAGGAAGGCUCUUGGAUAGAGUCGUGGGUGGUUCAGCC(aptamer) 48 132-B3 32nt, D-RNA GGAUCGCAGGGGCGUGGCUGGGGUGGGCGACCNOX-f 32nt (aptamer) 49 132-B3 33nt, D-RNAGGAUCGCAGGGGCGUGGCUGGGGUGGGCGAUCC NOX-f 33nt (aptamer)

It is within the scope of the present invention that, if no sequencesare explicitly given for the individual sections of the nucleic acidsaccording to the invention, these can be freely chosen according to thetechnical teaching disclosed herein, i.e. can be chosen so that theyexhibit the necessary binding behaviour to the respective targetmolecule and/or are able to form the structures, in particular secondarystructures, described herein.

Furthermore, it is within the scope of preferred embodiments of thepresent invention that in the case where, in sequences that areidentified as RNA sequences, T is given instead of U, then T shalldenote U.

The present invention is described in more detail hereinafter with theaid of the following Figures and Examples, which disclose furtherfeatures, embodiments and advantages. In this connection:

FIG. 1A shows aptamers generated by in vitro selection againstD-21AS-HMGA1a/b, which bind the 21AS-HMGA1a/b domain;

FIG. 1B is a representation of the identified, repeatedly occurringsequence regions of the aptamers generated by in vitro selection againstD-21AS-HMGA1a/b, which bind the 21AS-HMGA1a/b domain;

FIG. 2 is a sequence comparison of HMGA1a/b and HMGA2;

FIG. 3 is a shortening of HMGA1a/b-binding aptamer NOX-f;

FIG. 4 shows the binding properties of shortened HMGA1a/b-bindingaptamer NOX-f;

FIG. 5 shows a competition assay for measuring the binding of HMGA tothe double-strand natural target DNA in the multi-well plate assay; thebinding of the spiegelmer competes with the binding of the recombinantHMGA1b to the biotinylated dsDNA (AT hook motif). The detection of thebound HMGA1b is carried out through the His-Tag via nickel HRP, whichconverts a substrate into a fluorescing signal;

FIG. 6 shows a comparison of spiegelmer NOX-A and spiegelmer NOX-f (48nt; 33 nt) in the competitive multi-well plate assay; in the plateassay, spiegelmer NOX-A as well as spiegelmer NOX-f and its shortenedvariant spiegelmer NOX-f33 prevent the binding of recombinant HMGA1b toits naturally occurring binding partner in the low nanomolar range.

FIG. 7 shows the activity of 2 kDa-PEG-coupled spiegelmer NOX-A as wellas non-functional control spiegelmer in the competitive multi-well plateassay; the PEGylated spiegelmer NOX-A competes with the binding ofrecombinant HMGA1b to the AT hook motif of the dsDNA with an 1050 of 15nM; the inverse control spiegelmer of NOX-A shows at high spiegelmerconcentrations a non-specific interaction with HMGA1b;

FIG. 8 shows a western blot; detection of immobilised HMGA1b bybiotinylated spiegelmer; recombinant HMGA1b migrates in theelectrophoretic field like a 20 kDa large protein and can be recognisedat low concentration (3 nM) by the biotinylated spiegelmer (here withthe example of NOX-A); an inverse control spiegelmer could not recogniseHGMGA1b;

FIG. 9 shows the activity of free and PEGylated spiegelmer NOX-A in thecompetitive multi-well plate assay;

FIG. 10 is an investigation of the packing of PEGylated spiegelmer inmicelles in the “RiboGreen exclusion assay”;

FIG. 11 shows the stability of PEI spiegelmer micelles in the “RiboGreenexclusion assay”;

FIG. 12 shows the efficient uptake of spiegelmer packed in PEI micelles,in particular a comparison of the transfection of “naked” spiegelmercompared to spiegelmers packed in micelles, with the example of thespiegelmer NOX-A-3′PEG2 kDa.; the cells which have been transfected withspiegelmer micelles exhibited at a lower setting of the camerasensitivity (camera gain) a stronger fluorescence in the cytosolcompared with cells that had been incubated only with pure spiegelmer;the efficiency with both transfection methods is >95%;

FIG. 13 shows the release of spiegelmer from the endosomal compartment;spiegelmer micelles exhibited a significantly higher fluorescencecompared to pure spiegelmer; spiegelmer micelles exhibited a point-like,perinuclear, and also cytoplasmic distribution pattern; the point-likedistribution indicates a localisation in endosomal compartments; thediffuse distribution in the cytosol and on the plasma membrane indicatesspiegelmer released from endosomes;

FIG. 14 is a proliferation assay with “naked” spiegelmer; dose-dependentinhibition of the proliferation of MCF-7 cells at high spiegelmerconcentrations after 2 days in the cell culture medium (quantificationvia resazurin);

FIG. 15 shows the proliferation of H1299 cells (“non-small cell lungcancer”) after treatment with PEI packed NOX-A-2 kDa PEG; inhibition ofthe proliferation of H-1299 cells at 1 μM spiegelmer, applied asPEI-spiegelmer micelles (N/P 2.5); NOX-A showed a slight inhibition ofthe proliferation compared to the control spiegelmer;

FIG. 16 shows the inhibition of the HMGA1a/b-induced cdc25a geneexpression, detected by quantitative RT-PCR; determination of thespecific inhibition of the cdc25a mRNA expression in H-1299 cells by 1μM NOX-A spiegelmer micelles (N/P 2.5) by means of RT-PCR;

FIG. 17 shows the dose-dependent inhibition of the cdc25a mRNAexpression by spiegelmer NOX-A; quantification of the dose-dependentinhibition of the cdc25a mRNA expression in H1299 cells by means ofRT-PCR; NOX-A spiegelmer micelles (N/P 2.5) showed starting at 250 nM aspecific inhibition of the cdc25a mRNA expression; at a concentration >4μM a non-specific effect of the control spiegelmer was found, as well astoxic effects due to the polyethyleneimine (PEI) at >10 μM (data notshown);

FIG. 18 shows the inhibition of the tumour growth in the xenograft modelin naked mice by the spiegelmer NOX-A; inhibition of the tumour growthafter subcutaneous injection of PSN-1 cells by 2 mg/kg spiegelmermicelles (N/P 2.5). Spiegelmer NOX-A produced a significant reduction intumour growth;

FIG. 19 shows the statistical analysis of the data from the xenograftexperiment; inhibition of the tumour growth after subcutaneous injectionof PSN-1 cells by 2 mg/kg spiegelmer micelles (N/P 2.5); end pointanalysis and representation as box-and-whisker plot. NOX-A produced ahighly significant reduction of the tumour growth (p=0.0098 compared toPBS and p=0.022 compared to inverse control spiegelmer);

FIG. 20 shows the tissue distribution of spiegelmer NOX-A in thexenograft experiment; quantitative analysis of the distribution ofspiegelmer NOX-A in the plasma and tissues; a high concentration ofspiegelmer NOX-A could be detected in the tumour tissue, compared to theother tissues and plasma.

FIG. 21 shows tissue distribution of spiegelmer packed in micelles andunpacked spiegelmer, 24 and 96 hours after the last injection in thexenograft experiment; quantitative analysis of the distribution ofnon-functional spiegelmers in plasma and tissues; in the tumour tissue asignificantly raised concentration of spiegelmer could be detected inthe case of a spiegelmer packed in micelles compared to the othertissues and plasma, after 24 hours and 96 hours.

FIG. 22: shows distribution of spiegelmer packed in micelles andunpacked spiegelmer in plasma and in the tumour 24 and 96 hours afterthe last injection in the xenograft experiment; quantitative analysis ofthe distribution of a non-functional spiegelmer in plasma and tumour; inthe tumour tissue a significantly raised concentration of spiegelmercould be detected in the case of a spiegelmer packed in micellescompared to unpacked spiegelmer, after 24 hours and 96 hours.

EXAMPLE 1 HMGA1a/b-Binding Spiegelmers

1.1 HMGA1a/b-Binding Sequences

The HMGA1a/b-binding RNA spiegelmers were generated by in vitroselection against D-21AS-HMGA1a/b and subsequent shortening steps. Thegenerated aptamers, which bind the 21AS-HMGA1a/b domain, are shown inFIG. 1A.

1.1.1 Ranking and Aptamer Level

The different clones (see FIG. 1A) were prepared as aptamers (D-RNA) bymeans of standard phosphoramidite synthesis and were radioactivelylabelled at the 5′ end by kinasing (see below). The clones were thenanalysed as regards their affinity and activity by means of equilibriumbinding assay at two concentrations of D-bio-21aa HMGA1a/b.

Radioactive Labelling by Kinasing:

Substance [final] RNA 5 μM T4 forward reaction buffer (Invitrogen) 1x T4polynucleotide kinase (Invitrogen) 10 U/10 μl_(Reaction batch) [γ-³²P]-ATP 1 μl/10 μl_(Reaction batch)

The reaction ran for 1 hour at 37° C. and was then stopped by heating(10 minutes at 65° C.). The separation of radioactive nucleotides fromlabelled oligonucleotides was carried out by an analyticalpolyacrylamide gel electrophoresis (PAGE) (see hereinafter). A“crush-and-soak” gel elution was then carried out with ammonium acetateand precipitation with ethanol (see hereinafter). The amount of purifiedRNA was estimated from the radioactivity of the pellets (after theprecipitation) compared to the radioactivity of the cut-out strip.

Polyacrylamide Gel Electrophoresis (PAGE)

For the preparative purification of oligonucleotides, % to 2 volumes ofconcentrated sample buffer for denaturing PAGE were added to thereaction batches. In addition large-scale standards were prepared asnecessary (each 250 pmole) and taken up in sample buffer. The batcheswere denatured for 5 minutes at 95° C. and cooled on ice. A preparative,denaturing 7% or 10% PAA gel (200×200×1.5 mm) was preheated (ca. 1 hour)by applying a maximum voltage of 600 V at 40-50 W. After rinsing thecups with 1×TBE the samples were plotted. After completion of theseparation (50 minutes at 50 W) the gel was placed on a fluorescingthin-layer chromatography plate protected by transparent film (dye60F₂₅₄). The bands were visualised as shadows (“UV shadowing”) by meansof UV light (254 nm) and were cut out with a scalpel. A “crush-and-soak”gel elution with ammonium acetate was then performed.

“Crush-and-Soak” Gel Elution

To elute oligonucleotides from PAA gels, after comminuting the cut-outPAA gel strips 500 μl of ammonium acetate (2 M) was added using apipette tip or a spatula. The “crush-and-soak” elution was carried out2×1.5 hours at 68° C. in a thermoshaker (1000 rpm). The supernatantswere freed from gel residues by “Ultrafree-MC” small columns(Millipore/Amicon, Schwalbach, Germany) in a table centrifuge(16,100×g). The RNA eluted in this way was then desalted byprecipitation with ethanol.

Ethanol Precipitation

For the ethanol precipitation 1-2 μl of glycogen were used asprecipitation auxiliary. After adding 2.5 volumes of absolute ethanoland vortexing, the oligonucleotides were precipitated for 30 minutes at−80° C. and centrifuged off for 30 minutes at 16,100 g, 4° C. The pelletwas washed once with 70% ethanol and centrifuged for 5 minutes at 16,100g, 4° C.

Recording of Binding Isotherms in the Equilibrium Binding assay

2 pmole of each of the 5′ radioactively labelled aptamers were complexedin biotinyl-D-HMGA1a/b-21mer (EPSEVPTPKRPRGRPKGSKNK [Seq. ID. 17]; seeFIG. 2), produced by Bachem (Weil am Rhein, Germany). Solutions in theconcentration range 1-3000 nM (or for the two-point measurement with 300nM and 30 nM or 100 nM and 10 nM peptide) were incubated for 1 hour at37° C. in selection buffer (10 mM Tris HCl pH7.4, 5 mM KCl, 0.8 mMMgCl₂, 0.1% Tween). A solution without biotinylated D-HMGA1a/b-21merserved as background control. The peptide and complexes were thenimmobilised within 30 minutes at 37° C. with 10 μl of streptavidinUltraLink gel. The radioactivity of the suspension was measured. Thesupernatant was removed. The matrix was then washed once with 100 μl ofselection buffer and then precipitated with selection buffer. Bymeasuring the radioactivity the aptamer fraction present together withbiotinyl-D-HMGA1a/b-21mer in the complex was determined for each peptideconcentration. The dissociation constants of the active species and theproportion of active molecules were determined by graphical plotting andfit (GraFit, Version 4.0.10, Erithacus Software).

Results

For all clones synthesised as aptamers (D-RNA) a dissociation constantfor the binding to the 21 amino acid-long D fragment of HMGA1a/b(Biotinyl-D-HMGA1a/b-21mer) of 8-22 nM was determined in the equilibriumbinding assay (FIG. 1A).

1.1.2 Shortening in Example 132-B3

All selection candidates exhibited a repetitively occurring sequencemotif GGGCG or GGGUG or GGGAG, which is stabilised at the 5′ end and atthe 3′ end by a helix/stem motif (FIG. 3).

An analysis of the probable structure and precipitation of the RNAaptamers according to Zuker (Nucleic Acids Res. 2003 Jul. 1;31(13):3406-15) showed that the predetermined stem/helix structure hadlengthened in some cases (132-C3, 132-33, 132-C4, 132-E2, 132-A2,132-H1, 132-F1, 122-G2, 122-E2, see FIG. 1A). This stem-Helix structureformed the basis for the further shortening of these candidates. Thisfurther shortening of the candidates was carried out by identifying andstabilising the minimal binding motif by precipitation analysis followedby deletion analysis of the synthetic D-RNAs with respect to the bindingto the HMGA1a/b fragment. These binding properties were determined byequilibrium binding assay. FIG. 3 shows by way of example in thecandidate NOX-f (132-B3) the shortening of the aptamer on the basis ofthe stabilising stem structure, which can be found in lengthened form inthe candidates 132-C3, 132-B3, 132-C4, 132-E2, 132-A2, 132-H1, 132-F1,122-G2, 122-E2 (see FIG. 1A). A shortening to a 32 nucleotide-longaptamer variant of NOX-f with a 6 nucleotide-long stem (NOX-f 32 nt) didnot lead to any loss of the binding properties to the 21aa HMGA1a/bfragment (FIGS. 3 and 4). The artificial insertion of an adenosine atthe third position of the 5′-position stem led to a theoreticalformation of a 7 nucleotide-long stem without a looped-out region andserved to complete the stem in the 3′ region (NOX-f 33 nt, FIGS. 3 and4). The measurement of the binding properties (affinity and activity) bymeans of equilibrium binding assay on the 21 amino acid-long domain ofHMGA1a/b was not influenced by these changes.

The sequences 132-G2, 122-A1, 122-C1, 122-B2 and 122-B4 have on theother hand at the 5′ end and 3′ end of the repetitively occurringsequence motif (GGGCG or GGGUG or GGGAG) a significantly shorter stemstructure. A shortening of the stem structure led to a binding loss. Apossible shortening of the central region, which is longer for thesesequences, between the repetitive sequence motif (GGGCG or GGGUG orGGGAG) was not carried out.

The Seq. IDs of the aptamer sequences of the HMGA-binding nucleic acidsdisclosed herein are as follows:

Seq. ID Internal Reference RNA/Peptide 34 132-C3, NOX-h D-RNA (aptamer)35 132-B3, NOX-f (48 nt) D-RNA (aptamer) 36 132-C4 D-RNA (aptamer) 37132-E2 D-RNA (aptamer) 38 132-A2 D-RNA (aptamer) 39 132-H1, NOX-i D-RNA(aptamer) 40 132-F1 D-RNA (aptamer) 41 132-G2, NOX-g D-RNA (aptamer) 42122-A1, NOX-A D-RNA (aptamer) 43 122-C1, NOX-B D-RNA (aptamer) 44 122-B2D-RNA (aptamer) 45 122-E2, NOX-C D-RNA (aptamer) 46 122-G2, NOX-E D-RNA(aptamer) 47 122-B4, NOX-D D-RNA (aptamer) 48 132-B3 32 nt, NOX-f 32 ntD-RNA (aptamer) 49 132-B3 33 nt, NOX-f 33 nt D-RNA (aptamer)

As has already been discussed herein and is known to those skilled inthe art in this field the enantiomer, consisting of L-nucleotides, of anaptamer, i.e. of a D-nucleic acid which was generated against aD-peptide, binds to the mirror-image enantiomer of the D-peptide, i.e.the naturally occurring L-peptide. This L-nucleic acid is herein alsoreferred to as spiegelmer and otherwise exhibits in principle the samebinding properties as the aptamer.

1.2 Characteristic Properties of HMGA/b-Binding Spiegelmers 1.2.1Repetitive Sequence Elements: Box A1 and Box A2

A repetitive sequence element of the sequence GGGCG or GGGUG or GGGAG ischaracteristic of all spiegelmers that bind to HMGA1a/b. This sequenceelement appears twice in HMGA1a/b-binding spiegelmers (FIGS. 1A and 1B).The sequence element lying closer to the 5′ end of the spiegelmers isherein referred to as Box A1. The sequence element lying closer to the3′ end of the spiegelmers is on the other hand referred to herein as BoxA2. Box A1 and Box A2 and their mutual arrangement probably representthe decisive feature of HMGA1a/b-binding spiegelmers.

1.2.2 Sequence Section Between Box A1 and Box A2

Between Box A1 and Box A2 there is either a sequence section with alength of six to seven nucleic acids or 12 to 22 nucleotides (FIGS. 1Aand 1B). Since these sequence sections differ not only in their length,they are discussed separately.

Case 1: Sequence Section Comprises Six to Seven Nucleotides

If the sequence section lying between Box A1 and Box A2 has a length ofsix nucleotides, then the sequence section exhibits the sequence UGGUUG,UGGCUG, CGGUUG, AGGUUG or GUGUAA. An insertion of one nucleotide(uracil) into the sequence CGGUUG, which leads to the sequence CGGUUUG,has neither a negative nor a positive influence on the bindingproperties of the spiegelmers.

Case 2: Sequence Section Comprises 12 to 22 Nucleotides

If the sequence section lying between Box A1 and Box A2 has a length of12 to 22 nucleotides, then this sequence section comprises two sequenceregions of equal length, which can possibly hybridise with one another(Helix C). The hybridisation is in this case effected by in each casethree to six nucleotides. Three to five unpaired nucleotides are locatedbetween the nucleotides forming the Helix C. One to three nucleotidesare present unpaired between the 3′ end of Box A1 and the 5′ end ofHelix C. One to five nucleotides can be present unpaired between the 3′end of Helix C and the 5′ end of Box A2.

1.2.3 Helical Structure at the 5′ End and 3′ End of the Spiegelmers

All HMGA1a/b-binding spiegelmers are characterised at their 5′ and 3′ends by sequence sections which can hybridise with one another (Helix A1and Helix A2, (FIGS. 1A and 1B). The number of nucleotides hybridisingwith one another in each case can vary from four to eight. In thisconnection, this presumably double-strand region can extend to the 5′end of Box A1 and the 3′ end of Box A2. Should this not be the case,then Box A1 and Box A2 can be flanked by nucleotides that additionallyhybridise with one another (Helix B1 and Helix B2). This can involveregions of in each case four to eight nucleotides (FIGS. 1A and 1B).

Within the scope of the invention forming the basis of the presentapplication, various classes of nucleic acids and in particularL-nucleic acids which bind to the target molecule have been identified.The following illustration and description of these classes, which areherein also termed cases, is to this extent an integral part of thepresent invention. For each class their principal structure andexemplary L-nucleic acids for this class are specified hereinafter usingthe respective abbreviations of the L-nucleic acids.

Case 1: 132-C3, 132-B3, 132-C4, 132-E2, 132-A2, 132-H1, 132-F1, 122-G2,132-B3 32 nt, 132-B3 33 nt

N₁=U, C, A, G; N₂=G, U; N₃=U, C; N₄=U, A; N₅=G, A; N₆=G, A, U; N₇=G, U;

N₈=U or no nucleotide;N_(x)=zero to five nucleotides;N_(y)=zero or six nucleotides;N_(z)=zero to six nucleotides;

Helix A1 and Helix A2=in each case four to eight nucleotides, whichcompletely or partly hybridise with one another, in which the sum of thein each case mutually hybridising nucleotides of Helix A1 and Helix A2and Helix B1 and Helix B2 is 10 to 12 nucleotides;

Helix B1 and Helix B2=in each case four to eight nucleotides, whichhybridise with one another, in which the sum of the in each casemutually hybridising nucleotides of Helix A1 and Helix A2 and Helix B1and Helix B2 is 10 to 12 nucleotides.

The molecules are active also after the shortening at the 5′ end and atthe 3′ end. After the removal of the Helix A1 and A2 as well as theregions N₆N7 and GN_(y) the shortened molecules retain their bindingproperties. This was demonstrated for the shortened variants 132-B3 32nt (NOX-f 32 nt) and 132-B3 33 nt (NOX-f 33 nt) (see FIGS. 3 and 4).

Case 2A: 132-G2, 122-A1, 122-C1, 122-B2, 122-B4

N_(a)=one to five nucleotidesN_(b)=three nucleotidesN_(c)=three to five nucleotidesN_(d)=two to five nucleotidesN_(e)=one to two nucleotides, preferably A or UU

Helix A1 and Helix A2=in each case five to six nucleotides, whichcompletely or partly hybridise with one another,

=in each case five to six nucleotides, which hybridise with one another.

Case 2B: 122-E2

N_(i)=two nucleotides, preferably CAN_(j)=two nucleotides, preferably AGN_(c)=four nucleotides, preferably GAUG

Helix A1 and Helix A2=in each case six nucleotides, which hybridise withone another,

Helix B1 and Helix B2=in each case five nucleotides, which hybridisewith one another,

=in each case three nucleotides, which hybridise with one another.

EXAMPLE 2 Domain Approach 2.1 Determination of the Interaction ofHMGA1a/b Spiegelmers and Recombinant HMGA1b in the Competition AssayExecution/Method Cloning of His6-Labelled HMGA1b

The BD-Freedom™ ORF clone GH00552L1.0 (high mobility group AT hook1)with the sequence coding for HMGA1b was purchased from BioCatHeidelberg. The sequence had already been changed therein so that thestop codon is converted into a codon coding for leucine, in order topermit C-terminal fusions. The sequence of the clone correspondsgenerally to the sequence stored in the RefSeq data bank under No.NM002131. The sequence coding for HMGA1b was amplified by means of astandard PCR with the primers HMG_fwd1 (TCGACACCATGGGTGAGTC, Seq. ID 34)and HMG rev1 (GTCTAGAAAGCTTCCCAACTG, Seq. ID 35). In this connection thebase after ATG was changed from A to G, in order thereby to introduce aNcoI interface. The PCR product was cleaved according to themanufacturer's instructions with the restriction enzymes NcoI andHindIII (both from NEB, Frankfurt am Main, Germany) and purified via anagarose gel. The vector pHO2d (Fasshauer et al. (1997) J. Biol. Chem.272:28036-28041)) was similarly cleaved with NcoI and HindIII andpurified via an agarose gel. The Vector pHO2d permits the expression ofa protein fused to the C-terminal end with a sequence of six histidineresidues (His6-tag), under the control of a T7-promotor (Fasshauer etal., 1997, JBC 272:28036).

The purified and cleaved PCR product was ligated into the preparedvector overnight at 15° C. with the aid of a T4 ligase, corresponding tothe manufacturer's instructions (MBI Fermentas, St. Leon-Roth, Germany).Bacteria of strain DH5□ were transformed with the ligation product. Thecorrectness of the plasmids from obtained colonies was checked bysequencing. The fusion protein HMGA1b-His6 coded by pHO2d/HMGA1b has,compared to the natural HMGA1b protein, a glycine (G) instead of serine(S) at position 2, and after the C-terminal glutamine (Q) a leucine (L)(see above), followed by five further amino acids (G S L N S) (coded bythe vector), to which the six histidines (H) are joined.

Expression and Purification of HMG1b-His6

For the expression of the fusion protein bacteria of strain BL21 weretransformed with the plasmid pHO2d/HMGA1b. The expression of the fusionprotein was induced with isopropylthio-β-D-galactoside (IPTG). After 4hours the bacteria were centrifuged off for 15 minutes at 10,000×g andthe pellet was stored at −20° C. until further use.

For the extraction of the fusion protein 25 ml of extraction buffer (1%n-octyl-β,D-thioglucopyranoside (OTG) in 50 mM Na_(x)PO₄ buffer, pH 8.0,250 mM NaCl, 10 mM imidazole and MiniProtease inhibitor tablets (Roche,Mannheim, Germany) (5 hrs/50 ml)) were added to a frozen bacteria pelletfrom 500 ml of culture, followed by 5 μl of benzonase (gradel; MERCK,Darmstadt, Germany), homogenised by pipetting and pipetting off, andincubated for 5 min at RT. This was followed by centrifugation for 15mins at 10,000×g (RT). The supernatant was filtered through a flutedfilter and then added to a HIS-SELECT column (HIS-SELECT Cartridge,Sigma, Deisenhofen, Germany) equilibrated with wash buffer (50 mMNa_(x)PO₄ buffer, pH 8.0, 250 mM NaCl, mM imidazole, all from MERCK,Darmstadt, Germany). After washing the column with 10-15 ml of washbuffer the fusion protein was eluted with elution buffer (250 mMimidazole in wash buffer) in 0.5-1 ml size fractions. Protein-containingfractions were checked for purity by means of gel electrophoresis (16%polyacrylamide gel according to Schäger & Jagow, 1987, Anal. Biochem.166:368-379). Fractions with fusion protein were purified, if necessarydialysed using a suitable buffer, and after protein determination weretested again for purity. The purified fusion protein was stored inaliquots at −20° C.

Determination of the Interaction of HMGA1a/b Spiegelmers and HMGA1b-His6

A test based on the 96-well format was used for a more detailed analysisof the affinity of the HMGA1a/b-spiegelmers for HMGA1b. In this test thebinding of the HMGA1a/b spiegelmers to HMGA1b-His6 prevents itsinteraction with a DNA oligonucleotide that has a binding site forHMGA1a/b. This DNA oligonucleotide (dsDNA AT hook) (Fashena et al.,1992) is labelled on one strand with a biotin molecule, via which it canbe bound to plates coated with streptavidin. The detection ofHMGA1b-His6 bound to DNA is carried out with horseradish peroxidasemodified with nickel (Nickel-HRP), which transforms a fluorogenicsubstrate. In this assay the spiegelmer displaces the recombinant HMGA1bfrom its natural binding partner. On account of the 1:1:1 stoichiometryof spiegelmer/rHMGA1b/dsDNA AT Hook, a direct prediction can be maderegarding the affinity of the spiegelmers for HMGA1b. The principle ofthe assay is illustrated in FIG. 5.

To carry out this test spiegelmers in various concentrations andHMGA1b-His6 (0.36 μg/ml; ca. 30 nM) in a total volume of 100 μl areincubated for 10 minutes in a tapered floor plate at room temperaturewhile shaking. The incubation solution also contains: 25 mM Tris/HCl, pH7.0 (Ambion, Austin, Tex., USA), 140 mM KCl (Ambion, Austin, Tex., USA),12 mM NaCl (Ambion, Austin, Tex., USA), 0.8 mM MgCl2 (Ambion, Austin,Tex., USA), 0.25 mg/ml BSA (Roche, Mannheim, Germany), 1 mM DTT(Invitrogen, Karlsruhe, Germany), 18-μg/ml poly(dGdC) (Sigma,Deisenhofen, Germany)), 0.05% Tween 20 (Roche, Mannheim, Germany). 2 μlof biotinylated DNA oligonucleotides dsDNA AT hook (equimolar mixture of5′biotin-TCGAAAAAAGC CTGGC (34 nt) and 5′GCCAGTTTTTTTTTTTTTTTTTTGCTTTTTT(31 nt); 75 M in 150 mM NaCl (Ambion, Austin, Tex., USA)) are then addedand incubated for a further 10 mins at RT while shaking. The batches arethen transferred to a black 96-well plate coated with streptavidin(ReactiBind from Pierce, Bonn, Germany)) and incubated for 30 mins at RTwhile gently shaking. Following this the wells of the plate are washedthree times, each time with 200 μl of TBSTCM (20 mM Tris/HCl, pH 7.6(Ambion, Austin, Tex., USA); 137 mM NaCl (Ambion, Austin, Tex., USA), 1mM MgCl2 (Ambion, Austin, Tex., USA), 1 mM CaCl2 (Sigma, Deisenhofen,Germany), 0.05% Tween (Roche, Mannheim, Germany)). 50 μl of a dilutenickel-HRP solution are added to each well (ExpressDetector nickel-HRP,(Medac, Hamburg, Germany) 1:1000 in 10 mg/ml BSA (Roche, Mannheim,Germany) in TBSTCM) and incubated for hour at RT while gently shaking.The wells are then washed again three times with 200 μl TBSTCM eachtime. 100 μl of the fluorogenic HRP substrate (QuantaBlue, Pierce, Bonn,Germany) are then added to each well and the fluorescence is measuredafter 15 mins (ex: 340/em: 405 nm).

Result

It was shown that the spiegelmers NOX-A (50 nt), NOX-f (33 nt) and NOX-f(48 nt) compete in a concentration-dependent manner with the binding ofHMGA1b-His6 to the biotinylated DNA-Oligonucleotide (FIG. 6). A IC50 ofca. 15 nM is found for spiegelmer NOX-A.

In contrast to the active spiegelmer, a control spiegelmer with ainverse sequence to NOX-A showed in a concentration of up 0.5 μM noeffect on the binding of HMGA1b-His6 to the DNA oligonucleotide, andnon-specific interactions with HMGA1b-His6 occur only at concentrationsabove 1 μM (FIG. 7).

2.2 Use of Spiegelmers to Detect HMGA1b by Western BlotExecution/Methods

The recombinantly expressed HMGA1b was separated by gel electrophoresison a 16% PAA-tricin gel and transferred by means of electroblotting tonitrocellulose membranes. The membrane was then blocked with 5% skimmedmilk and 100 nM non-specific spiegelmer in 1×TBST (20 mM Tris/HCl pH7.6, 137 mM NaCl, 0.1% Tween) for 1 hour and washed three times for 10minutes with 1×TBST. The detection of the recombinant HMGA1b was carriedout with spiegelmer NOX-A biotinylated at the 5′ end (5′bioNOX-A).5′bioNOX-A was diluted in 1×TBST with 1 mM each of calcium and magnesium(TBST+Ca/Mg) and 100 nM non-specific spiegelmer and incubated for 1.5hours. The blot was then washed three times for 10 minutes with1×TBST+Ca/Mg and the bound biotinylated spiegelmer was incubated with ananti-biotin antibody in TBST+Ca/Mg for 45 minutes. The blot was thenwashed five times for 10 minutes with 1×TBST+Ca/Mg and the secondaryantibody coupled with horseradish peroxidase (HRP) was detected by meansof LumiGLO detection reagent (Cell Signaling Technology).

Result

The binding of a 5′-terminal biotinylated spiegelmer to therecombinantly expressed HMGA1b was demonstrated by means of theaforedescribed process. Similarly to a detection based on antibodies, 5μg of HMGA1b were detected with 3 nM bio-NOX-A after transfer to a blotmembrane. The inverse spiegelmer of NOX-A could not recognise HMGA1b,which confirms the specific binding of NOX-A (FIG. 8).

EXAMPLE 3 PEI-Spiegelmer Formulation 3.1 Principle of thePolyethyleneimine-Mediated Transfection of Spiegelmers

The target molecule HMGA1a/b is expressed in the cytosol and finds astranscription factor its natural binding partner, the double-strand DNAin the cell nucleus. The HMGA1a/b-mediated cellular responses should beantagonised by binding of the spiegelmer to HMGA1a/b in the cytosol, andcompetition of the HMGA1a/b bound by the AT hooks to the DNA in cellnucleus. On account of the negative charge of the plasma membrane DNAand RNA molecule are not readily taken up by passive transport from acell. One of the approaches to the intracellular transport by nucleicacids is the condensation or packing with charged particles or reagents,resulting in a charge of the overall complex. This complex is easilytaken up through endocytosis and thus passes into the cytosol of thecell. Disadvantages of this method are the stability of the DNA/RNA andthe release of the nucleic acid from the endosomal compartment. In thecytosol of the cell a lysosome is quickly formed from the constrictedendosome by the introduction of proteases or nucleases and byprotonation of the compartment. Nucleases digest the nucleic acids thereand in addition the nucleic acid is not stable in the acidic medium. Thewhole complex is rapidly transported again out of the cell by exocytosisand decomposition in the Golgi apparatus, and therefore only a fewnucleic acids pass into the cell. One of the preconditions for asuitable transfection system is thus the stabilisation as well as therelease of the nucleic acid from the endosome into the cytosol. Asregards stability RNA spiegelmers have ideal properties for atransfection of eukaryotic cells, since being unnatural enantiomers theyare not cleaved by enzymes.

The selected transfection system is based on the formation of micellesof nucleic acids with branched polyethyleneimine (PEI). The phosphatebackbone of the nucleic acids interacts with the free nitrogen positionsof the PEI and forms small micelles by cross-branching, which have apositive charge on account of the PEI. In this connection PEI with amolecular weight of 3 to 800 kDa is used. The smaller the PEI, thesmaller are the formed micelles. The use of 25 kDa cross-branched PEI(Sigma-Aldrich Cat. No. 40; 872-7) leads on addition of nucleic acids tothe formation of polyplexes of size 100 nm up to 500 nm, thoughtypically to polyplexes of size 100 to 200 nm. As a rule anitrogen/phosphate ratio of 2:1 to 5:1 is used, in some cases even up to20:1. The packing of the nucleic acid in micelles results in a change ofthe zeta potential of the complex to ˜(+)21 mV with a N/P ratio of 3. Itis known that with increasing, positive zeta potential of complexes thetoxicity to culture cells rises. These micelles are however easily takenup as endosomes by a cell by constriction of the plasma membrane. ThePEI now buffers inflowing protons, as a result of which many chlorideions in the interior of the endosome lead to a swelling of thecompartment on account of the osmotic pressure. This effect of PEI isdescribed in the literature as the proton sponge effect (Sonawane etal., JBC, 2003, Vol. 278; No. 45(7) pp. 44826-44831) and ultimatelyleads to the rupture of the endosome and to the release of thespiegelmers into the cytosol.

The nucleic acid-PEI complex has a tendency on account of a stronglypositive charge to interaction and aggregation with serum proteins, andalso to exhibit the aforedescribed cell toxicity. Thus, it has beendescribed in the literature that high doses of nucleic acid-PEI micellesafter subcutaneous and intravenous injection in rats can rapidly lead toan accumulation in the lungs and thus to embolisms/infarcts. Thesolution to this problem is to derivatise the nucleic acid with 2 kDapolyethylene glycol (PEG). These residues surround the micelles like ashield and prevent the binding to serum proteins (Ogris et al., GeneTherapy, 1999, 6(595-605). Furthermore, the zeta potential is reduced to+/−0 mV, which leads to a lower cell toxicity while retaining the buffercapacity of the PEI as regards the proton sponge effect.

3.2 Spiegelmer Activity with PEG2000

The lead candidates NOX-A and NOX-f were produced synthetically asaptamer and spiegelmer with a 3′-terminal amino group, and were thenPEGylated via the amino radical. It was shown by means of equilibriumbinding assays that PEGylation has no influence on the bindingproperties of the aptamers to the HMGA1a/b fragment. Furthermore, it wasshown by means of competition assays with recombinant full-lengthHMGA1a/b that also the binding of spiegelmers to the full-lengthHMGA1a/b is independent of the 3′-terminal PEGylation (FIG. 9).

3.3 Spiegelmer Packing

The packing of sterile, PEGylated spiegelmer was carried out in PBS byadding 25 kDa of cross-branched polyethyleneimine (PEI) (ALDRICH, Cat.:40,872-7) in a ratio of the absolute nitrogen fraction of the PEI to theabsolute phosphate of the ribonucleic acid backbone of 2.5:1 (N/P 2.5).The sterile, autoclaved PEI solution had a concentration of 200 mM freenitrogen groups and was adjusted to a pH of 7.4 with 1 M hydrochloricacid. The sterile filtered spiegelmer was taken in a concentration of upto 700 μM in 1×PBS with Ca/Mg and after addition of sterile filtered PEIwas incubated for 30-60 minutes at room temperature. Ideally the complexformation takes place with the smallest possible adjusted concentrationof added spiegelmer, since high concentrations of spiegelmer lead torandomly large aggregates. The formation of spiegelmer micelles wasmeasured by means of a dye exclusion assay. For this, it was determinedhow much spiegelmer can be detected by the dye before and after packingin micelles. RiboGreen (M. Probes) was used as dye, and the fluorescencewas measured with an ELISA reader. 1 μM spiegelmer was added in eachcase to 100 μl 1×PBS and increasing amounts of PEI were added. 100 μl of0.2 μg/μl RiboGreen were placed in a 96-well microtitre plate suitablefor fluorescence, and after incubating the micelle batch for 30 minutesat room temperature 10 μl were pipetted into the microtitre plate.Starting from a N/P ratio of 2, more than 90% of the spiegelmers werepresent as micelles (FIG. 10). In this connection PEI alone had noinfluence on the fluorescence of the dye.

3.4 Stability of Spiegelmer Micelles

1 μM of spiegelmer micelles were stored under conditions specified inFIG. 11. The stability of spiegelmer micelles was measured by the dyeexclusion assay described in Section 3.3 A stability study of themicelles showed that the storage of micelles in different media as wellas at different temperatures has no influence on the spiegelmermicelles. The freeze drying of ribozyme/PEI complexes without any lossof the properties of the ribozyme is also described in the literature(Brus-C et al., J. Control Release, 2004, Feb., 20, 95(1), 199-31).

3.5 Uptake of Spiegelmer Micelles

The intracellular uptake of spiegelmer micelles was established in acell culture system of HS578T cells. 1×10⁴ HS578T cells were allowed togrow on sterile 20 mm size cover classes to a confluence of 30-40%.5′-labelled spiegelmer NOX-A-3′-PEG was packed with a N/P ratio 2.5:1 inmicelles, added in a concentration of 1 μM to the cells, and incubatedfor 16 hours at 37° C. As control for the passive uptake of spiegelmers,1 μM of pure fluorescence-labelled spiegelmer was in each case incubatedwith the cells. The cells were then washed three times with 1 ml of PBSand fixed for 30 minutes with 3% paraformaldehyde. The preparations wereagain washed three times with 1 ml of PBS, incubated for a further 10-20seconds with a DAPI solution (1 μl stock to 10 ml 1×PBS) to stain thechomatin in the cell nucleus, washed once more, and covered with amounting solution. The preparations prepared in this way were analysedin a fluorescence microscope (emission 488 nm/extinction 514-522 nm).

It was shown that spiegelmer micelles have a higher transfection ratecompared to “naked”, unpacked spiegelmers (FIG. 12)

The transfection efficiency was in this connection >95% of all cells andhad no influence on the morphology of the cells. The 5′-FITC-coupledspiegelmer was mainly to be found in the cytosol and associated with theplasma membrane. The point-like distribution indicates an inclusion incompartments and the diffuse pattern points to released spiegelmer. Onlya slight spiegelmer signal could be detected in the cell nucleus.

3.6 Release of Spiegelmer

The point-like distribution of the spiegelmer in the cytosol andperinuclear space of the H578T cells points to an accumulation incompartments of the cells, for example endosomes. To check the releaseof the spiegelmers from these compartments the distribution pattern ofindividual, greatly enlarged cells was analysed (FIG. 13). In additionto the point-like localisation of the spiegelmers, a diffusedistribution pattern in the cytosol and on the plasma membrane wasdetected, which points to the endosomal release of the spiegelmers. Thispattern was not found in the case of “naked” spiegelmers.

EXAMPLE 4 Bioactivity In Vivo

4.1 Proliferation Assay without PEI

Effect on the Proliferation of MCF-7 Cells

The potential role of HMGA1a/b in cell division was investigated bymeans of proliferation assays. First of all spiegelmer was added in ahigh dose as “naked” nucleic acid to the cell culture medium and thegrowth of the cells was followed over time The breast cancer cell lineMCF-7 was used as model, since in these cells a smaller (antagonising)expression of HMGA1a/b was found, and the role of HMGA1a/b in theproliferation of these cells had already been described in theliterature. Reeves et al. (Reeves-R et al., Molecular and CellularBiology, January 2001, p 575-594) showed that the over-expression ofHMGA1a/b in MCF-7 cells leads to an increased proliferation, and theinhibition of HMGA1a/b by means of expressed antisense constructsinhibits the proliferation of MCF-7 cells.

Execution/Method

0.5×10⁴ MCF-7 cells (ATCC) were seeded out in 96-well plates (Costar)with a flat, transparent base and cultured for 16-24 hours in 100 μlRPMI 1640 medium with 10% foetal calf serum (FCS). The cells were thenwashed with PBS and cultured for a further 48 hours with standard cellculture medium with the direct addition of sterile filtered spiegelmer.This was followed by the addition of 10 μl of a resazurin solution (0.44mM in PBS) to the respective batches and further incubation for 2 hoursat 37° C. The transformation of resazurin by the cell metabolismcorrelates directly with the number of cells. The change in colour wasmeasured in a Fluostar Optima multidetection plate reading device (BMG)(emission 544 nm, extinction 590 nm). Each value was determined threetimes per experiment and referred to the values of untreated controlcells.

Result

NOX-A inhibited after two days in a dose-dependent manner theproliferation of MCF-7 cells (n=12) (FIG. 14). The maximum inhibition ofthe proliferation to ca. 30% of the value of untreated cells was foundat 40 μM. At concentrations up to 40 μM no non-specific effect of theinverse control spiegelmer was found.

4.2 Proliferation Assay with PEI

Effect of Spiegelmer Micelles on the Proliferation of H-1299 CellsExecution/Method

1×10⁴ NCI-H-1299 cells (lung carcinoma cells; ATCC) were seeded out in24-well plates (Costar) with a flat, transparent base and cultured for16-24 hours in 1 ml RPMI 1640 medium with a 10% FCS. The cells were thenwashed twice with PBS and cultured for a further three days with cellculture medium containing 1% FCS and spiegelmer micelles. The packing ofsterile, PEGylated spieglemer was carried out beforehand in PBS byadding 25 kDa cross-branched polyethyleneimine (PEI) (Sigma) in a ratioof the absolute nitrogen fraction of the PEI to the absolute phosphateof the ribonucleic acid backbone of 2.5:1 (N/P 2.5). The sterilespiegelmer was used in a concentration of 30 μM and after the additionof the PEI was incubated for 30-60 minutes at room temperature. Thespiegelmer micelles were then diluted to 1 μM with cell culture mediumcontaining 1% FCS, added directly to the washed cells, and incubated forthree days at 37° C.

This was followed by addition of 100 μl resazurin solution to therespective batches and further incubation for 2 hours at 37° C. Thetransformation of resazurin by the cell metabolism correlates directlywith the number of cells. 100 μl were removed from the batches,transferred to a 96-well plate, and the colour change was measured in aFluostar Optima multidetection plate reading device (BMG) (emission 544nm, extinction 590 nm). Each value was determined twice per experimentand referred to the values of untreated control cells.

Result

The use of PEI (N/P 2.5) with 1 μM spiegelmer did not initially have anyeffect on cell proliferation. By reducing the amount of FKS in the cellculture medium to below 1% it was shown that the transfection withspiegelmer micelles has an influence on the proliferation of H-1299cells, which was not previously visible with 10⁹6 FKS (FIG. 15).Possibly FKS stimulates the proliferation to such an extent that theslight effect could not be observed. The reduction of the FKSconcentration in MCF-7 cells lead to the death of the cells over aperiod of 3 days.

4.3 Inhibition Tumour Marker cdc25a (with PEI)

Effect on the HMGA1a/b-mediated regulation of cell cycle factors, in theexample of the potential oncogene cdc25a.

Reeves et al. (Molecular and Cellular Biology, January 2001, p 575-594)showed by means of cDNA arrays through over-expression of HMGA1a/b inMCF-7 cells that HMGA1a/b induces the expression of a large number ofgenes. At the same time cell cycle factors and growth factors such asfor example cdc25a, identified as a potential oncogene (cell divisioncycle 25a phosphotase), which plays a decisive role in the control ofthe transition from the G1 phase to the S phase of the cell cycle, areover-expressed by a factor of up to 100. The activation of such controlpoints leads after inhibition of the cell cycle progression either tothe transcription of genes which are involved in DNA repair or, if theDNA damage is irreparable, to the induction of apoptosis. As cellculture test system H-1299 cells were chosen for this purpose, sincethey have already exhibited an increased expression of HMGA1a/b.

Execution/Method

1×10⁴ H-1299 cells were seeded out in 24-well plates (Costar) with aflat, transparent floor and cultured for 16-24 hours in RPMI 1640 mediumcontaining 10% FCS (volume 1 ml). The cells were then washed twice withPBS and cultured for a further three days in cell culture medium withspiegelmer micelles containing 10% FCS. The packing of sterile,PEGylated spiegelmer was carried out beforehand in PBS by adding 25 kDacross-branched polyethyleneimine (PEI) (Sigma) in a ratio of theabsolute nitrogen fraction of the PEI to the absolute phosphate of theribonucleic acid backbone of 2.5:1 (N/P 2.5). The sterile spiegelmer wasused in a concentration of 30 μM and, after adding PEI, was incubatedfor 30-60 minutes at room temperature. The spiegelmer micelles with cellculture medium containing 1% FCS were then diluted to the respectiveconcentration, added directly to the washed cells, and incubated forthree days at 37° C. The cells were washed twice with PBS and harvestedby means of a cell scraper. The mRNA of the cells was then isolated fromthe cells by means of Roti-Quick-Kits (Roth, Cat. No. 979.1) and 0.2-1μg of total RNA was used as template for the PCR of cdc25a and GAPDH.

The primers for the amplification of GAPDH were as follows: forwardprimer: 5′-ACATGTTCCAATATGATTCC-3′ and reverse primer:5-TGGACTCCACGACGTACTCAG-3′ at an annealing temperature of 51° C., andfor the amplification of cdc25a: forward primer:5′-GAGGAGTCTCACCTGGAAGTACA-3′ and reverse primer5′-GCCATTCAAAACCAGATGCCATAA-3′ at an annealing temperature of 59° C. ThePCR conditions were as follows: 0.2-0.75 μM primer, 1.5 mM MgCl2 and 0.2mM dNTPs. Every two PCR cycles an aliquot of 5 μl was quantified byPicoGreen and evaluated by correlation with GAPDH as load control: forthis, in the first step for each investigated sample the so-called“crossing point” value (CP) of the reference gene is subtracted from theCP value of the gene being investigated (dCP=CP target gene minus CPreference gene). CP is defined as the number of PCR cycles that arerequired in order to reach a constantly defined fluorescence value. Thesame amount of newly synthesised DNA is found at the CP in all reactionvessels. After this standardisation the dCP value of a control (in thiscase GAPDH) is subtracted from the dCP value of the experimentallytreated samples; one arrives at the so-called “delta-delta CT”calculation model. The relative expression difference of a samplebetween the treatment and the control (ratio), normalised to thereference gene and referred to a standard sample, is found from thearithmetic formula 2^(−ddCP).

dCP=CP(cdc25a)−CP(GAPDH)

ddCP=dCP(treatment spiegelmer NOX-A)−dCP(control: PBS or NOX-A inverse)

Ratio=2^(−ddCP)

Result

cdc25a and HMGA1a/b were detected in MCF-7 and H-1299 cells by means ofRT-PCR. MCF-7 cells showed with a low expression of HMGA1a/b also a lowexpression of cdc25a, whereas HMGA1a/b and cdc25a were stronglyexpressed in H-1299 cells. The transfection of H-1299 cells for two dayswith HMGA1a/b-binding spiegelmers led to a significant, dose-dependentreduction of the expression of cdc25a mRNA (FIG. 16 and FIG. 17).

Up to a concentration of 4 μM a control speigelmer exhibited nonon-specific effect, neither on the GAPDH nor on the cdc25a mRNAexpression. From this it can be concluded that the HMGA1a/b-inducedover-expression of the potential oncogene cdc25a can be inhibited bymeans of spiegelmers.

EXAMPLE 5 Effectiveness Study: Xenograft Model Effect of Spiegelmers onTumour Growth In Vivo

In order to test the hypothesis that HMGA1a/b-binding spiegelmersinhibit the growth of tumours in vivo, a xenograft model was developedfor the strongly HMGA1a/b-expressing pancreatic carcinoma cells PSN-1.On the basis of this model a therapeutic experiment was carried out with2 mg/kg NOX-A spiegelmer micelles at a N/P of 2.5 (see Example 3,paragraph 3.3).

Execution/Method

Male naked mice (NMRI: nu/nu) (group size n=8) were subcutaneouslyinjected in the side with in each case 10⁷ PSN-1 cells (ECACC) and thetumour growth was observed over 22 days. The animals had a mean weightof 25-27 g and were 6-8 weeks old. The active spiegelmer NOX-A-3′PEG andthe inverse control spiegelmer in INV-3′PEG were packed in micelles asdescribed above by adding PEI in a N/P ratio of 2.5. 100 μl of thespiegelmer micelle suspension (corresponding to 3.46 nmole/animal or 2mg/kg) were in each case subcutaneously injected daily into the vicinityof the tumour. The tumour volume and bodyweight were measured threetimes a week. The animals were sacrificed on day 22 and the distributionof NOX-A in the plasma, liver, kidneys and tumour was quantified.

For this purpose the tissues were homogenised in hybridisation buffer(0.5×SSC pH 7.0; 0.5% (w/v) SDSarcosinate) and centrifuged for 10 minsat 4000×g. The supernatants obtained were stored at −20° C. untilfurther use.

The amount of spiegelmer in the plasma samples and in the tissuehomogenates was investigated by means of a hybridisation assay (Droletet al. (2000) Pharm. Res. 17:1503). The hybridisation assay is based onthe following principle: the spiegelmer to be detected (L-RNA molecule)is hybridised on an immobilised L-DNA oligonucleotide probe (=captureprobe NOX-A; in this case: 5′-CCCATATCCACCCACGTATCAGCCTTTTTTTT-NH2-3′;complementary to the 5′ end of HMGA1a/b-NOX-A) and detected with abiotinylated detection L-DNA probe (=detector probe NOX-A; in this case:5′-biotin-TTTTTTTTGGCTGAAACCACCCACATGG-3′; complementary to the 3′ endof HMGA1a/b-NOX-A). For this purpose a streptavidin alkaline phosphataseconjugate is in a further step bound to the complex. After adding achemiluminescence substrate light is generated and measured in aluminometer.

Immobilisation of the oligonucleotide probe: 100 μl of the capture probe(0.75 pmole/ml in coupling buffer: 500 mM Na₂HPO₄, pH 8.5, 0.5 mM EDTA)were transferred to each well (depression in a plate) in DNA-BIND plates(Corning Costar) and incubated overnight at 4° C. The probe was thenwashed three times with 200 μl of coupling buffer each time andincubated for 1 hour at 37° C. with in each case 200 μl of blockingbuffer (0.50 (w/v) BSA in coupling buffer). After washing again with 200μl of coupling buffer and 3×200 μl hybridisation buffer the plates canbe used for the detection.

Hybridisation and detection: 10 μl EDTA plasma or tissue homogenate weremixed with 90 μl of detection buffer (2 pmole/μl of detector probe inhybridisation buffer) and centrifuged. Further purifications werecarried out as necessary. The batches were then denatured for 10 mins at95° C., transferred to the suitably prepared DNA-BIND wells (see above)and incubated for 45 mins at ca. 40° C. The following wash steps werethen carried out: 2×200 μl hybridisation buffer and 3×200 μl 1×TBS/Tween20 (20 mM Tris-Cl pH 7.6, 137 mM NaCl, 0.10 (v/v) Tween 20). 1 μlstreptavidin alkaline phosphatase conjugate (Promega) was diluted with 5ml of TBS/Tween 20. 100 μl of the diluted conjugate were added to eachwell and incubated for 1 hour at room temperature. The following washsteps were then carried out: 2×200 μl TBS/Tween 20 and 2×200 μl of assaybuffer (20 mM Tris-Cl pH 9.8, 1 mM MgCl₂). 100 μl of CSPD “Ready-To-UseSubstrate” (Applied Biosystems) were then added, incubated for 30 minsat room temperature, and the chemiluminescence was measured in aFluostar Optima multidetecton plate reading device (BMG).

Result

In a preliminary experiment it was shown that H-1299 cells aftertransplanting as a tumour grew significantly more slowly than PSN-1, andon comparing the individual animals exhibited an inhomogeneous tumourgrowth and therefore appeared unsuitable as xenograft model for atreatment study. PSN-1 cells exhibited an aggressive tumour growthwithin 22 days. It was shown that NOX-A nicelles at a dose of 2 mg/kgreduced the growth of PSN-1 tumours significantly compared to the PBScontrol (FIG. 18). The weight of the animals was unaffected by thetreatment with spiegelmer micelles. The control spiegelmer did notexhibit any non-specific inhibition of the tumour growth and likewisehad no effect on the weight of the animals. The differences in tumoursizes were, from day 10 of the treatment with NOX-A3′PEG micelles,significant or highly significant compared to untreated animals (PBScontrol (student's t-test). The end point analysis after 22 days showeda highly significant, specific reduction in tumour growth (p=0.0098compared to PBS and p=0.022 compared to inverse control spiegelmer)(FIG. 19). Mice treated with PBS showed an average tumour growth of 2.5cm³, animals treated with controlled spiegelmer had an average tumourvolume of 2.6 cm³ and animals treated with NOX-A had an average tumourvolume of 1.2 cm³ after 22 days (box-and-whisker analysis). Thiscorresponds to a reduction of the tumour growth of more than 50%.

The analysis of the tissue distribution of NOX-A showed a highconcentration in the tumour (FIG. 20).

EXAMPLE 6 Comparison of the In Vivo Tissue Distribution of Packed andUnpacked Spiegelmer

In order to check the efficient incorporation of spiegelmer micelles, anon-functional spiegelmer (Proof Of Concept=POC) was PEGylated at the 3′end with PEG 2 kDa and packed with a nitrogen/phosphate ratio (N/P) of2.5 in micelles (see Example 3, paragraph 3.3). In a similar way to theprotocol described in Example 5, this approach was adopted forspiegelmer packed in micelles as well as for free, unpacked spiegelmer.

Execution/Method

Male naked mice (NMRI: nu/nu) (group size n=8) were in each caseinjected subcutaneously in the side with 10⁷ PSN-1 cells (ECACC) and thetumour growth was observed over 25 days. The animals had a mean weightof 25-27 g and were 6-8 weeks old. The non-functional spiegelmerPOC-3′PEG was packed in micelles by adding PEI in a N/P ratio of 2.5, asdescribed above. Spiegelmer POC-3′PEG not packed in micelles served ascontrol for the incorporation not mediated by PEI. 100 μl of thespiegelmer-micelle suspension or spiegelmer solution (corresponding to1500 nmole/kg and 2000 nmole/kg) were injected daily subcutaneously intothe vicinity of the tumour. The tumour volume and body weight weremeasured three times a week. 24 and 96 hours after the last injectiontwo animals from each group were sacrificed and the distribution ofPOC-3′PEG (packed/unpacked) in the plasma, brain, heart, lungs, liver,kidneys, gallbladder, pancreas and tumour was quantified.

For this purpose the tissue was homogenised in hybridisation buffer(0.5×SSC pH 7.0; 0.50 (w/v) SDSarcosinate) and centrifuged for 10 minsat 4000×g. The resultant supernatants were stored at −20° C. untilfurther use.

The amount of spiegelmer in the plasma samples and in the tissueshomogenates was investigated by means of a hybridisation assay (Droletet al. (2000) Pharm. Res. 17:1503). The assay is based on the followingprinciple: the spiegelmer (L-RNA molecule) to be detected is hybridisedon an immobilised L-DNA oligonucleotide probe (=capture probe POC; here:5′-NH2(C7)-TTTTTTTTTAGCTCTGCACAGCGCT-3′; complementary to the 3′ end ofPOC) and is detected with a biotinylated detection L-DNA probe(=detector probe POC; here:5′-CCGCATCAGACCGAGTTTCCTTATTTTTTTT-Biotin-3′; complementary to the 5′end of POC). For this, a streptavidin alkaline phosphatase conjugate wasbound in a further step to the complex. After addition of achemiluminescence substrate, light is generated and measured in aluminometer.

Immobilisation of the oligonucleotide probe: 100 μl of the POC captureprobe (0.75 pmole/ml in coupling buffer: 500 mM Na₂HPO₄ pH 8.5, 0.5 mMEDTA) were transferred to each well (depression in a plate) in DNA-BINDplates (Corning Costar) and incubated overnight at 4° C. The probe wasthen washed three times with 200 μl of coupling buffer and incubated for1 hour at 37° C. with 200 μl of blocking buffer (0.5% (w/v) BSA incoupling buffer). After washing again with 200 μl of coupling buffer and3×200 μl of hybridisation buffer, the plates can be used for thedetection.

Hybridisation and detection: 10 μl of EDTA plasma or tissue homogenatewere mixed with 90 μl of detection buffer (2 pmole/μl POC detector probein hybridisation buffer) and centrifuged. Further purifications werecarried out as necessary. The batches were then denatured for 10 mins at95° C., transferred to the suitably prepared DNA-BIND wells (see above),and incubated for 45 mins at ca. 40° C. The following wash steps werethen carried out: 2×200 μl of hybridisation buffer and 3×200 μl1×TBS/Tween 20 (20 mM Tris-Cl pH 7.6, 137 mM NaCl, 0.1% (v/v) Tween 20).1 μl of streptavidin alkaline phosphatase conjugate (Promega) wasdiluted with 5 ml of 1×TBS/Tween 20. 100 μl of the dilute conjugate wereadded to each well and incubated for one hour at room temperature. Thefollowing wash steps were then carried out: 2×200 μl of 1×TBS/Tween 20and 2×200 μl of 1× assay buffer (20 mM Tris-Cl pH 9.8, 1 mM MgCl₂). 100μl of CSPD “Ready-To-Use Substrate” (Applied Biosystems) were thenadded, incubated for 30 mins at room temperature, and thechemiluminescence was measured in a Fluostar Optima multidetection platereading device (BMG).

Result

The analysis of the weight distribution of the non-functional spiegelmerPOC-3′PEG, which was packed in micelles, showed after 24 hours asignificantly higher concentration in the tumour tissues(24.925+/−13.301 pmole/mg) compared to the unpacked spiegelmer(0.840+/−0.255 pmole/mg) (FIG. 21 A). Whereas the concentration of thepacked spiegelmer had halved (11.325+/−7.050 pmole/mg) after a furtherthree days (96 hours), only a very small amount of the unpackedspiegelmer could be detected (0.120+/−0.057 pmole/mg).

The plasma level of unpacked spiegelmer (2.950+/−0.438 pmole/ml) after24 hours was comparable to that of the PEI-packed spiegelmer(1.930+/−2.729 pmole/ml). After 96 hours clear differences were found,in which about four times the amount of packed spiegelmer compared tothe unpacked spiegelmer was detected.

A slight accumulation in the kidneys was observed after 24 hours forboth formulations, whereas a slight accumulation in the liver andgallbladder was found only for unpacked spiegelmer. After 96 hours, forboth formulations only minor amounts of spiegelmer were detected in theliver and kidneys. On the other hand, slightly raised values were foundin the gallbladder and pancreas (but with a high standard deviation) forpacked spiegelmer compared to unpacked spiegelmer.

To summarise, compared to the weight distribution (24 and 96 hours afterthe last injection) of spiegelmers in the presence and absence of PEI,it was found that spiegelmer micelles have a significantly prolongedresidence time in the plasma and tumour compared to unpacked material(FIG. 21B) and thus represent a promising approach to the use ofspiegelmers directed against intracellular target molecules.

The following citations are incorporated herein by way of reference.

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The features of the invention disclosed in the preceding description,claims and drawings can be essential both individually as well as in anycombination for the implementation of the invention in its variousembodiments.

1-139. (canceled)
 140. A composition comprising an L-nucleic acid thatbinds an intracellular target molecule and a delivery vehicle.
 141. Thecomposition according to claim 140, which intracellularly delivers saidL-nucleic acid.
 142. The composition according to claim 140, whereinsaid delivery vehicle is selected from the group consisting of aconjugate and a physical means.
 143. The composition according to claim140, wherein said delivery vehicle is selected from the group consistingof a liposome, a nanoparticle, a microparticle, a dendrimer and acyclodextrin.
 144. The composition of claim 140, wherein said deliveryvehicle comprises a vesicle comprising a polypeptide, apolyethyleneimine (PEI), an amphipathic molecule or combinationsthereof.
 145. The composition according to claim 140, wherein saiddelivery vehicle comprises a conjugate, wherein said conjugate comprisesa fusogenic peptide, a receptor that mediates endocytosis, a signalpeptide or a lipophilic molecule.
 146. The composition according toclaim 140, wherein said delivery vehicle comprises electroporation,iontophoresis, pressure, ultrasound or shock waves.
 147. The compositionaccording to claim 144, wherein said PEI comprises branches, and saidPEI comprises a molecular weight of about 25 kDa.
 148. The compositionaccording to claim 144, wherein said PEI forms a micelle or amicelle-like structure.
 149. The composition according to claim 140,wherein said L-nucleic acid comprises a spiegelmer.
 150. The compositionaccording to claim 149, wherein said spiegelmer comprises amodification.
 151. The composition of claim 150, wherein saidmodification comprises polyethylene glycol (PEG).
 152. The compositionaccording to claim 151, wherein said PEG comprises a molecular weight ofabout 1,000 to 10,000 Da; about 1,500 to 2,500 Da; or about 2,000 Da.153. The composition according to claim 144, wherein the ratio ofnitrogen groups of the PEI to phosphate groups of the L-nucleic acid isabout 1 to 20; about 1.5 to 10; about 2 to 5; or about 2 to
 3. 154. Thecomposition according to claim 140, wherein said intracellular receptorcomprises a polypeptide, a carbohydrate, a nucleic acid, a lipid orcombinations thereof.
 155. The composition according to claim 140,wherein said intracellular receptor comprises a transcription factor oran architectonic transcription factor.
 156. A pharmaceutical compositioncomprising the composition according to claim 140, and apharmaceutically acceptable carrier.
 157. A method for binding anintracellular receptor comprising: providing a cell comprising at leastone intracellular receptor, providing the composition of claim 140, andincubating the cell with the composition, wherein the L-nucleic acid ofsaid composition binds the intracellular receptor in the cell, whereinthe intracellular receptor is selected from the group consisting of acarbohydrate, a polypeptide, a nucleic acid, a lipid and combinationsthereof.
 158. The method according to claim 157, wherein said L-nucleicacid comprises a spiegelmer.
 159. The method according to claim 157,wherein said intracellular receptor is selected from the groupconsisting of a transcription factor and an architectonic transcriptionfactor.
 160. A method of manufacturing a medicament, comprisingcombining the composition of claim 140 and a pharmaceutically acceptablecarrier
 161. The method of claim 160, wherein said cell is obtained froman animal or a human comprising a disease associated with saidintracellular receptor.
 162. The method of claim 161, wherein saiddisease is selected from the group consisting of a tumour disease, avirus infection and arteriosclerosis.
 163. The method of claim 162,wherein said tumour disease is selected from the group consisting of amesenchymal tumour, an epithelial tumour, a benign tumour, a malignanttumour and a metastasizing tumour.